United States
Environmental Protection
Agency
Research and Development
Uptake,
Metabolism, and
Disposition of
Xenobiotic
Chemicals in Fish
Wisconsin Power
Plant Impact Study
^
EP 600/3
80-082
LIBRARY
»m PHOTK
-DISC'S.
-------
RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development. U S Environmental
Protection Agency have been grouped into nine series These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields
The rime series are
1 Environmental Health Effects Research
2 Environmental Protection Technology
3 Ecological Research
4 Environmental Monitoring
5 Socioeconomic Environmental Studies
6 Scientific and Technical Assessment Reports (STAR)
7 Interagency Energy-Environment Research and Development
8 ' Special' Reports
9 Miscellaneous Reports
This report has been assigned to the ECOLOGICAL RESEARCH series This series
describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials Problems are assessed for their long- and short-term influ-
ences Investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects This work provides the technical basis
for setting standards to minimize undesirable changes in living organisms in the
aquatic, terrestrial, and atmospheric environments
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161
-------
EPA-600/3-80-082
August 1980
UPTAKE, METABOLISM AND DISPOSITION OF XENOBIOTIC CHEMICALS IN FISH
Wisconsin Power Plant Impact Study
by
John Lech
Mark Melancon
Department of Pharmacology and Toxicology
Medical College of Wisconsin
Milwaukee, Wisconsin
Grant No. R803971
Project Officer
Gary E. Glass
Environmental Research Laboratory-Duluth
Duluth, Minnesota
This study was conducted in cooperation with
Wisconsin Power and Light Company,
Madison Gas and Electric Company,
Wisconsin Public Service Corporation,
Wisconsin Public Service Commission, and
Wisconsin Department of Natural Resources
ENVIRONMENTAL RESEARCH LABORATORY-DULUTH
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
DULUTH, MINNESOTA 55804
,,.; * i \ V
.l*a ;.i V A
-------
DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory-
Duluth, U.S. Environmental Protection Agency, and approved for publication.
Approval does not signify'that the contents necessarily reflect the views
and policies of the U.S. Environmental Protection Agency, nor does mention
of trade names or commercial products constitute endorsement or recommen-
dation for use.
ii
-------
FOREWORD
The U.S. Environmental Protection Agency (EPA) was created because of
increasing public and governmental concern about the dangers of pollution to
the health and welfare of the American people. Polluted air, water, and land
are tragic testimony to the deterioration of our natural environment. The
complexity of that environment and the interplay between its components
require a concentrated attack on the problem.
Research and development, the necessary first steps, involve definition
of the problem, measurements of its impact, and the search for solutions.
The EPA, in addition to its own laboratory and field studies, supports
environmental research projects at other institutions. These projects are
designed to assess and predict the effects of pollutants on ecosystems.
One such project, which the EPA has supported through its Environmental
Research Laboratory in Duluth, Minnesota, is the study "The Impacts of
Coal-Fired Power Plants on the Environment." This investigation, carried out
by the Institute for Environmental Studies of the University of Wisconsin-
Madison, in cooperation with the Wisconsin Power and Light Company, Madison
Gas and Electric Company, Wisconsin Public Service Corporation, Wisconsin
Public Service Commission, and Wisconsin Department of Natural Resources, is
monitoring and evaluating the impacts of a new coal-fired power plant on the
immediate environment.
During the next year reports from this study will be published as a
series within the EPA Ecological Research Series. These reports will include
topics related to chemical constituents, chemical transport mechanisms,
biological effects, social and economic effects, and integration and
synthesis.
This report presents results from the Hazardous Chemicals in Fish
subproject. In a series of laboratory studies the investigators have
explored the effects and fate in fish of various chemical compounds that are
associated with fossil fuels.
Norbert A. Jaworski
Director
Environmental Research Laboratory
Duluth, Minnesota
iii
-------
ABSTRACT
The effects and fate in fish of a number of chemicals, including
hydrocarbons and chlorinated hydrocarbons, have been examined. The inter-
actions between these chemicals and fish (generally rainbow trout) have been
studied using several approaches: examination of the uptake, metabolism,
and elimination of selected chemicals by fish; assessment of the effects of
selected chemicals (inducing agents) on hepatic xenobiotic metabolizing
enzymes (assayed in vitro); and studies of the effects of inducing agents on
the metabolism and disposition of other chemicals in vitro.
The uptake and elimination of ltfC-labeled naphthalene, 2-methylnaph-
thalene, 1,2,4-tri-chlorobenzene, pentachlorophenol, and pentachloroanisole
were studied. Each of these chemicals was taken up rapidly by rainbow trout.
The C derived from all compounds except pentachloroanisole was released
rapidly (ti/2 of elimination < 1 day), whereas pentachloroanisole-derived
1!*C was released more slowly (ti/2 about 6 days). Increasing the duration
of exposure to llfC-naphthalene or 1'*C-2-methylnaphthalene affected the
elimination of llfC-containing components from these fish, apparently because
of a slower release rate for metabolites of these compounds which accumulated
in tissues during longer exposure periods.
Activities of cytochrome P-450-related xenobiotic metabolizing enzymes
in rainbow trout livers were induced (elevated by 3-methylcholanthrene-type
inducers but not by phenobarbital-type inducers). When fish that were
preinjected with 3-methylcholanthrene-type inducers were subsequently
exposed to llfC-labeled naphthalene, 2-methylnaphthalene, and trichloro-
benzene, the quantities of biliary metabolites in these fish were consider-
ably higher than those found in non-induced trout.
An inhibitor of cytochrome P-450-related xenobiotic metabolism,
piperonyl butoxide, was shown to reduce levels of biliary metabolites of
pentachloroanisole and di-2-ethylhexyl-phthalate in rainbow trout exposed to
these chemicals and to increase tissue levels of these chemicals.
The high levels of biotransformation products of these chemicals found
in fish bile during and after exposure to the chemicals in these studies
support the possible use of bile sampling in pollutant-modeling programs.
Most of the funding for the research reported here was provided by the
U.S. Environmental Protection Agency. Funds were also granted by the
University of Wisconsin-Madison, Wisconsin Power and Light Company, Madison
Gas and Electric Company, Wisconsin Public Service Corporation, and Wisconsin
Public Service Commission. This report was submitted in fulfillment of
iv
-------
Grant No. R803971 by the Environmental Monitoring and Data Acquisition Group,
Institute for Environmental Studies, University of Wisconsin-Madison, under
the partial sponsorhsip of the U.S. Environmental Protection Agency. The
report covers the period of July 1975-July 1978, and work was completed as
of April 1979.
-------
CONTENTS
Foreword iii
Abstract iv
Figures viii
Tables xil
Acknowledgment xv
1. Introduction 1
2. Conclusions and Recommendations 2
Conclusions 2
Recommendations 3
3. Overview 5
Objectives 5
Scope and limits of the investigation 5
Organic pollutants 6
Metabolic transformation of xenobiotics by fish 7
Accumulation and elimination of xenobiotics 10
Methodology 11
4. Results 23
Hepatic xenobiotic metabolizing activity in rainbow
trout 23
Studies of the fate of organic pollutants in fish .... 60
Effect of inducers on disposition of organic chemicals
in rainbow trout 122
5. Significance and Potential Applications of the Research .... 130
References 131
vii
-------
FIGURES
Number Page
1 Scheme for fractionation of rainbow trout liver 14
2 Distribution of marker enzymes from trout liver 27
3 Distribution of mitochondrial and microsomal marker enzymes from
trout liver 29
4 The effect of various inducing agents on selected microsomal
enzyme activities 31
5 Time-course for induction of arylhydrocarbon (benzo[a]pyrene)
hydroxylase by various polycyclic hydrocarbons 32
6 Protein dependency for various monooxygenations in rainbow
trout hepatic microsomes 35
7 Dose-response relationship for Aroclor 1242 36
8 Dose-response relationship for Firemaster BP6 37
9 Time-course of induction by Aroclor 1242 39
10 Time-course of induction by Aroclor 1254 40
11 Time-course of induction by Firemaster BP6 41
12 Hemoprotein P-450 difference spectra 43
13 Dose-response relationship for 3-naphthoflavone induction of
benzo[a]pyrene hydroxylation in rainbow trout 45
14 Lineweaver-Burk plots for ethoxycoumarin-0-deethylation by
rainbow trout hepatic microsomes 46
15 Lineweaver-Burk plots for ethoxycoumarin-0~deethylation by
rainbow trout hepatic microsomes with expanded ordinate to
demonstrate biphasic nature 48
16 Lineweaver-Burk plots for ethoxyresorufin-0-deethylation by
rainbow trout hepatic microsomes 49
17 Type I substrate binding spectra of hexobarbital and piperonyl
butoxide with rainbow trout hepatic microsomes 52
viii
-------
Number Page
18 Type II ligand binding spectrum of imidazole with rainbow trout
hepatic microsomes 53
19 Formation of metabolite-ferrocytochrome P-450 complex in rainbow
trout hepatic microsomes 55
20 Time-course of phenobarbital in trout liver 56
21 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of
microsomes from variously pretreated rainbow trout 61
22 Enlargement of the 40,000- to 60,000-dalton region of samples D,
E, and F from Figure 21 62
23 Tissue levels of 1!*C during exposure to ^C-haphthalene (0.005
mg/liter) and subsequent eliminations 63
24 Tissue levels of lkC during exposure to ^C-naphthalene (0.023
mg/liter) and subsequent eliminations 64
25 Tissue levels of 1!*C during and after multiple exposures of trout
to l^C-naphthalene (0.005 mg/liter) 65
26 Tissue levels of ll*C in trout during a 27-day exposure to
^C-naphthalene and subsequent elimination 67
27 Tissue levels of llfC in trout during a 26-day exposure to lkC-2-
methylnaphthalene and subsequent elimination 68
28 TLC profiles of tissue radioactivity from rainbow trout exposed
to 0.5 mg/liter :"^-naphthalene or ^'*C-2-methylnaphthalene for
24 h 71
29 Thin-layer chromatography of biliary llfC from carp exposed to
0.337 mg of lltC-2-methylnaphthalene/liter for 24 h 77
30 TLC of biliary 11+C from carp and sheepshead exposed to 1'*C-2-
methylnaphthalene 78
31 Time-course of PCP and PGA in several tissues of rainbow trout . . 79
32 Elimination of lf*C from PCP- and PCA-exposed rainbow trout .... 81
33 14C in blood, bile, and fat of rainbow trout expos'ed to 1IfC PCP
and lkC PGA for 4 and 8 h 82
34 Thin-layer radiochromatogram of samples prepared from tissues of
rainbow trout exposed to llfC-PCP 84
ix
-------
Number
35 Thin-layer radiochromatogram of samples prepared from tissues
of rainbow trout exposed to ItfC-PCA 85
36 Flow diagram for the isolation and purification of biliary DEHP
and metabolites 91
37 Thin-layer chromatography of fractionated rainbow trout bile ... 93
38 Effect of 3-glucuronidase hydrolysis on the TLC mobility of
Metabolites BI, BII and Bill 95
39 Thin-layer chromatography of Metabolites BI, BII and Bill after
incubation with 3-glucuronidase 96
40 Thin-layer chromatography of Metabolite BII, methylated following
B-glucuronidase hydrolysis 97
41 Thin-layer chromatography of Fraction D (top graph), and of the
acidic ether extract of this fraction after (3-glucuronidase
hydrolysis (hydrolyzed Metabolites DI), lower graphs, in two
solvents 98
42 Pathways for DEHP metabolism by rainbow trout 102
43 Metabolite patterns following incubation of llfC-DEHP with trout
liver homogenate 104
44 Influence of time on metabolism of ^C-DEHP by trout liver
mitochondrial and microsomal fractions 106
45 Influence of DEHP concentration on metabolism of ^C-DEHP by trout
liver mitochondrial and microsomal fractions 107
46 Distribution of marker enzymes and DEHP-metabolizing enzymes in
trout liver homogenate fractions 110
47 Effect of PBO on the metabolism of DEHP by trout liver
homogenates 114
48 Chemical structures of di-2-ethylhexylphthalate, 2 ,4,-dichloro-
phenoxyacetic acid-n-butyl ester, paraoxon and methylenedioxy-
phenyl compounds 119
49 Uptake and elimination of 1'fC-2-methylnaphthalene derived material
in rainbow trout 123
50 Thin-layer radiochromatographic profile of 2-methylnaphthalene
metabolites in trout bile 124
-------
Number Page
51 TLC of biliary 1
-------
TABLES
Number Page
1 Distribution of Hydrolytic Enzymes of Rainbow Trout Liver 24
2 Enzymatic and Protein Analyses of Subcellular Fractions of
Rainbow Trout Liver 25
3 Distribution of Microsomal Enzymes of Rainbow Trout Liver 26
4 Cytochrome P-450 Content of Variously-Induced Trout Hepatic
Microsomes 33
5 In vitro Effect of 3-Naphthoflavone on Arylhydrocarbon
(Benzo[a]pyrene) Hydroxylase Activity of Hepatic Microsomes
in Control Trout 34
6 Liver:Body Weight Ratios and Yields of Microsomal Protein 38
7 Cytochrome P-450 Content and 455:430 Peak Ratios of EtNC
Spectra 42
8 Microsomal Yields and Liver:Body Ratios After Pretreatment of
Rainbow Trout with Various Inducing Agents 47
9 Induction of Monooxygenation in Rainbow Trout Hepatic Microsomes
Following Intraperitoneal Pretreatment 47
10 Effect of Inducers on the Kinetics of Monooxygenation in
Rainbow Trout Hepatic Microsomes Following Intraperitoneal
Pretreatment 50
11 In Vitro Effects of a-Naphthoflavone and Metyrapone on
Arylhydrocarbon (Benzo[a]pyrene) Hydroxylase in Hepatic Microsomes
of Variously-Pretreated Rainbow Trout 51
12 Cytochrome P-450 Content and 455:430 Peak Ratios of EtNC
Spectra 51
13 Effect of Potential Inducing Agents upon Benzo[a]pyrene
Hydroxylation in Control Trout Hepatic Microsomes In Vitro .... 57
14 Typical Hepatic Microsomal Monooxygenase Activities of Rat and
Mouse Found in Authors' Laboratory 57
xii
-------
Number Page
15 Elimination Half-Lives of 1>*C from Fingerling Rainbow Trout
Exposed to ll+C-Naphthalene in Water on a Short-Term Basis .... 66
16 Elimination Half-Lives of lltC from Fish Exposed to Aqueous
^C-Naphthalene or llfC-2-Methylnaphthalene for Several Weeks . . 69
17 Uptake and Elimination of llfC-2-Methylnaphthalene by Carp After
Exposure to 0.013 mg 1'*C-2-Methylnaphthalene/Liter 72
18 Uptake and Elimination of 1'*C-2-Methylnaphthalene by Bluegill
Sunfish After Exposure to 0.013 mg ll*C-2-Methylnaphthalene/
Liter 72
19 Fraction of llfC in Muscle from Trout Exposed to 1 ^C-Naphthalene
or 1'*C-2-Methylnaphthalene Present as Polar Compounds 74
20 Biliary 14C Following Exposure of Fish to 1!*C-Naphthalene or
JltC-2-Methylnaphthalene 76
21 Half-Life (ti/2) of Pentachlorophenol and Pentachloroanisole
in Rainbow Trout Tissues 80
22 The GC/MS and TLC Analysis of Tissue Extracts from Rainbow Trout
Exposed to ^C-Pentachlorophenol 83
23 Effect of Piperonyl Butoxide on Distribution of 1!*C in Bile of
Rainbow Trout Exposed to ^C-Pentachloroanisole 87
24 GC/MS Analysis of Chromatographically-Separated ^C from
3-Glucuronidase-Treated Bile from Rainbow Trout Exposed to
''C-Pentachloroanisole 87
25 Uptake and Elimination of llfC-l,2,4-Trichlorobenzene by Rainbow
Trout: Short-Term Exposure 88
26 Uptake and Elimination of ^C-l^^-Trichlorobenzene by Rainbow
Trout: Long-Term Exposure 89
27 Elimination of Trichlorobenzene by Rainbow Trout 90
28 Tissue Levels of DEHP (and/or Metabolites) Following 24 h
Exposure to Aqueous DEHP at an Initial Level of 0.5 PPM 92
29 |3-Glucuronidase Hydrolysis of Major Bile Metabolite Fractions . . 94
30 Distribution of 1!*C in Fractionated Trout Bile 99
31 Gas Chromatographic-Mass Spectral Analysis of Phthalate
Metabolism 100
xiii
-------
Number Page
32 DEHP and Metabolites in Rainbow Trout and Catfish 101
33 Metabolism of DEHP by Trout Liver Homogenates 103
34 Metabolism of DEHP by Subcellular Fractions of Trout Liver
Homogenates and by Trout Blood Serum 105
35 Metabolism of 2,4-DBE by Subcellular Fractions of Trout Liver
Homogenates and by Trout Blood Serum 109
36 Hydrolysis of DEHP by Recombined Trout Liver Homogenate
Fractions Ill
37 Effect of Piperonyl Butoxide on Metabolism of DEHP and 2,4-DBE
by Trout Liver Subcellular Fractions and Serum 115
38 Effect of Piperonyl Butoxide on Metabolism of DEHP by Trout
Liver Homogenate and Blood Serum 116
39 Metabolism of DEHP by 2,000 g Supernatant of Liver Homogenate
from Control Trout and Trout Preexposed to Piperonyl Butoxide . . 117
40 Effect of Piperonyl Butoxide on Accumulation of 1!*C-DEHP in
Various Tissues of Rainbow Trout In Vivo 117
41 Effect of Piperonyl Butoxide on Accumulation of DEHP and MEHP
in Muscle of Rainbow Trout In Vivo 117
42 Effect of Microsomal Inhibitors on DEHP Hydrolysis In Vitro . . . 120
43 Effect of Microsomal Inhibitors on DEHP Metabolism In Vitro . . . 121
44 Effect of Pre-Administration of (3-Naphthof lavone on the
Disposition and Metabolism of ^C-Labeled Chemicals in Rainbow
Trout 125
xiv
-------
ACKNOWLEDGMENT
The authors wish to acknowledge the other researchers of the Department
of Pharmacology and Toxicology at the Medical College of Wisconsin who have
participated in the work included in this report. These contributors
include Clifford R. Elcombe, Andrew H. Glickman, Charles N. Statham,
Lawerence A. Menahan, Anthony Wu, Jill Say bolt, and Susan B. Szyjka.
xv
-------
SECTION 1
INTRODUCTION
The operation of a coal-fired power plant presents opportunities for a
variety of organic chemicals to reach the environment. When a power plant
is situated adjacent to a river or a lake, some of these organic pollutants
may be presented to fish inhabiting the general area of the power plant.
Although the levels of these chemicals in the water may be too low to result
in lethality or other obvious forms of toxicity to the fish, other more
subtle interactions may occur. In fact, the accumulation of such chemicals
by fish might be harmful not to fish themselves but to humans who consume
them.
Many organic chemicals are likely to be taken up from the aqueous
environment by fish and there may be considerable differences in the fate of
these chemicals within the fish. Among the most obvious are differences in
the biotransformation of these pollutants by the fish, differences in the
distribution of these pollutants or their biotransformation products in the
fish, and differences in the rates of elimination of these pollutants and
their biotransformation products by the fish. In addition, one chemical
might affect any of these parameters relative to another chemical in the
fish. For example, such interactions could result from induction or
inhibition of hepatic xenobiotic metabolizing enzymes.
Accordingly, the present study was designed to evaluate the uptake,
biotransformation, distribution, and elimination of selected organic
chemicals by fish and the ability of various chemicals to affect these
parameters.
-------
SECTION 2
CONCLUSIONS AND RECOMMENDATIONS
CONCLUSIONS
The characterization of rainbow trout liver subcellular fractions
showed that cytochrome(s) P-450 and most xenobiotic metabolizing enzymes are
present in the microsomal fraction, as is the case in higher animals.
Although total cytochrome P-450 levels were increased only slightly by 3-
methylcholanthrene-type inducers (g-naphthoflavone, Aroclors 1242 and 1254,
etc.)> a variety of cytochrom P450 related enzyme activities were increased
as much as 40-fold. Gel electrophoresis demonstrated the appearance of a
new cytochrome P-450 having a molecular witht of about 57,000 in response to
these inducers.
Studies with C-labeled compounds i,n vivo showed that naphthalene, 2—
methylnaphthalene, pentachlorophenol, pentachloroanisole, trichlorobenzene,
and di-2-ethylhexyl phthalate were taken up readily by fingerling rainbow
trout. After exposures to aqueous naphthalene, 2-methylnaphthalene,
pentachlorophenol, or trichlorobenzene for 1 day or less, the half-times for
elimination from various tissues were less than 1 day. Similar exposures to
pentachloroanisole resulted in half-times of elimination of about 1 week.
After long-term exposures of fingerling rainbow trout to aqueous naphthalene
or 2-methylnaphthalene, however, there was a slower rate of elimination of
at least part of the accumulated C, apparently due to a slower release
from certain tissues of biotransformation products than of the parent
chemicals.
Biotransformation products of these compounds were found in the bile of
the trout exposed to each chemical. Di-2-ethylhexyl phthalate and
pentachlorophenol were metabolized to the greatest extent, naphthalene and
2-methylnaphthalene somewhat less, and pentachloroanisole and
trichlorobenzene the least.
g-naphthoflavone, an inducer of xenobiotic metabolism, increased the
levels of biotransformation products of naphthalene, 2-methyl-naphthalene,
and trichlorobenzene found in trout bile and decreased the tissue levels of
the parent chemicals in rainbow trout exposed to these chemicals. Piperonyl
butoxide, an inhibitor of xenobiotic metabolism, decreased the levels of
biotransformation products of di-2-ethylhexyl phthalate and penta-
chloroanisole in trout bile and increased tissue levels of the parent
chemicals in rainbow trout exposed to these chemicals.
-------
RECOMMENDATIONS
We have found biotransformation products of the various chemicals under
study in fish tissues and bile. The accumulation of such biotransformation
products may be as important as the presence of the parent chemicals
themselves. These biotransformation products might be more toxic than the
original pollutants, and analysis for the original pollutants might miss the
bulk of the pollutant-derived material. Examples of metabolic
transformations that result in the formation of more toxic compounds are the
formation of proximate carcinogens from precarcinogens and the oxidation of
the weakly toxic parathion to the highly toxic paraoxon. An example of a
chemical whose metabolites are more likely to be found than the original
chemical is malathion, which is so rapidly metabolized that 24 h after
exposure only hydrolysis products are found in fish tissues. Additional
studies should be done in fish on the accumulation of the biotransformation
products of the various organic pollutants that arise from the operation of
a coal-fired power plant.
The high levels of the biotransformation products of various chemicals
found in fish bile are important for another reason. The use of bile may
provide a convenient method of sampling fish for determining previous
exposure of the fish to pollutants. Although some pollutants (particularly
lipophilic ones such as PCBs) accumulate to high levels in fish tissues, we
have shown that the metabolites of a wide variety of chemicals (including
relatively water soluble ones) accumulate to high levels in fish bile. In
some cases these metabolites may be found in bile at high levels days after
the exposure to the pollutant has been terminated. Work should continue on
the development of a pollution monitoring system based on the presence of
biotransformation products of organic pollutants in fish bile.
Because of the possibility that one chemical might affect the
metabolism and disposition of other chemicals in the environment, long-term
exposures of fish to likely environmental levels of such chemicals should be
performed to permit assessment of the effects of such exposures on fish
liver xenobiotic metabolizing enzymes.
In addition, studies should be undertaken to determine which organic
chemicals actually reach the aqueous environment from the operation of a
coal-fired power plant. Selections should be done in fish on the
accumulation of the biotransformation products of the various organic
pollutants that arise from the operation of a coal-fired power plant.
The high levels of the biotransformation products of various chemicals
found in fish bile are important for another reason. The use of bile may
provide a convenient method of sampling fish for determining previous
exposure of the fish to pollutants. Although some pollutants (particularly
lipophilic ones such as PCBs) accumulate to high levels in fish tissues, we
have shown that the metabolites of a wide variety of chemicals (including
relatively water soluble ones) accumulate to high levels in fish bile. In
some cases these metabolites may be found in bile at high levels days after
the exposure to the pollutant has been terminated. Work should continue on
-------
the development of a pollution monitoring system based on the presence of
biotransformation products of organic pollutants in fish bile.
Because of the possibility that one chemical might affect the
metabolism and disposition of other chemicals in the environment, long-term
exposures of fish to likely environmental levels of such chemicals should be
performed to permit assessment of the effects of such exposures on fish
liver xenobiotic metabolizing enzymes.
In addition, studies should be undertaken to determine which organic
chemicals actually reach the aqueous environment from the operation of a
coal-fired power plant. Selection of chemicals for the current studies with
fish was based mainly on the results of laboratory studies concerning the
extractability of components of coal and petroleum products into water.
-------
SECTION 3
OVERVIEW
OBJECTIVES
Briefly stated, the objective of this project is to investigate in fish
the dynamics of the uptake, biotransformation, and disposition of selected
hazardous chemicals which might arise from the operation of a coal-fired
power plant. In addition to simply measuring the uptake and release of
various hazardous chemicals by fish, the research centers on the
biotransformation and disposition of selected xenobiotic chemicals in fish
and encompasses three interrelated long-term objectives. The first
objective is to determine the metabolic pathways of potentially hazardous
chemicals in fish and the extent to which biotransformation influences the
level and persistence of these chemicals and their metabolites in fish
tissues. The second objective is to determine the role of biotransformation
in the protection of fish against toxic chemicals in the aquatic
environment. The third objective, which is implicit in these research
goals, is to evaluate the modification of biotransformation and excretion
processes in fish by chemicals such as polychlorinated biphenyls (PCBs),
polycyclic aromatic hydrocarbons (PAHs), and methylene-dioxyphenyl
synergists which are known to modulate xenobiotic metabolism in mammals.
The studies have been designed to investigate in fish the dynamics of
the uptake, biotransformation, distribution, and elimination of selected
hydrocarbons which are present in coal and petroleum and chlorinated
hydrocarbons which may be used in or derived from algacide treatment of
power-plant cooling systems. Data obtained from these studies will be used
to evaluate a pharmacokinetic model to predict the accumulation and
elimination of hazardous residues in fish exposed to spills or effluent
mixing zones arising from chlorination practices.
SCOPE AND LIMITS OF THE INVESTIGATION
In order to meet the objectives of the investigation, the following
studies have been initiated:
1. Short term and long term uptake, distribution, and elimination of
selected organic chemicals and their biotransformation products on
fish.
2. Xenobiotic metabolism by fish -in vivo.
3. Xenobiotic metabolism by fish in vivo.
-------
4. Modification of xenobiotic metabolism by fish in vitro*
5. Effects of such modification of xenobiotic biotransformation
processes in fish on the biotransformation, disposition, and
elimination of xenobiotics in vivo*
ORGANIC POLLUTANTS
For many years the hazards of waterborne chemicals were evaluated by
relatively insensitive criteria, such as whether the pollutants were harmful
to fish or other aquatic species. Experience with one class of organic
chemicals, the PCBs, has shown that some species of fish can accumulate
these chemicals to very high levels without apparent harm. These
accumulated PCBs, however, may have harmful effects on higher animals,
including man, which consume these fish. Information concerning which
pollutants are accumulated by fish, where they are stored, and in what form
they are stored, is germaine to rational approaches to monitoring for
potentially hazardous chemicals in water.
The composition of coal and petroleum and their wide-spread uses
suggest that these materials could be major contributors of organic
chemicals to the aquatic environment. Many organic chemicals appear in
water shaken with pulverized coal (Carlson and Caple 1979) or with various
oils (Boylan and Tripp 1971, Lee et al. 1974, Larson and Weston 1976).
Naphthalene and methylnaphthalenes are major constituents of these aqueous
extracts. Anderson (1975) utilized dispersions of crude oil and fuel oil in
water to study the uptake of petroleum constituents by several marine
species. Several invertebrates accumulated naphthalene and
methylnaphthalene, and killifish accumulated substantial amounts of
naphthalene.
Treatment of the cooling systems of power plants to prevent the buildup
of algae may contribute chlorinated organic pollutants either directly by
the use of pentachlorophenol or indirectly by chlorination.
Carlson and Caple (1979) have shown that several organic chemicals are
chlorinated by dilute chlorine under laboratory conditions. This process
also occurs during the chlorination of drinking water and sewage.
Laboratory studies of the chlorination of cooling-water concentrates under
conditions simulating those used in the cooling system of an electric power-
generating plant showed that 0.5% of the chlorine present became
incorporated into the organics present (Jolly et al. 1976). Except for
pesticides containing chlorine and PCBs, little has been done in studying
chlorinated hydrocarbons in fish.
Based on the above considerations, we selected for study naphthalene,
2-methylnaphthalene, pentachlorophenol, pentachloroanisole, and
1,2,4-trichlorobenzene. Although not a potential pollutant from power
plants, di-2-ethylhexylphthalate, a widespread ester pollutant, was already
under study when this project began, and this work has been completed.
-------
METABOLIC TRANSFORMATION OF XENOBIOTICS
A variety of species can modify the structures of chemicals that are
not constituents of or participants in the usual metabolic pathways. A
subcellular structure called the endoplasmic reticulum has the capability of
enzymically catalyzing the alteration of the structures of a wide variety of
organic compounds. In most animal species studied, the ability to catalyze
such reactions resides mainly in the liver, but to a lesser degree the
process occurs in a large variety of tissues. When liver tissue is
homogenized, this activity is associated with small particles called
microsomes. More specifically, this activity is due to the catalytic
activity of a class of hemoproteins, the cytochrome(s) P-450, so called
because of an absorption peak at about 450 nm. This enzyme activity, in
general, is called monooxygenase activity because of the activation of a
single oxygen atom from 02 during the course of the reaction. Although a
wide variety of reactions are catalyzed by cytochrome(s) P-450, the concern
of this study is those reactions related to xenobiotic chemicals. In
general, the foreign chemical is metabolized to a more polar compound that
is more readily eliminated by the organism. For example, one or more
hydroxyl groups may be added to a foreign chemical, increasing its water
solubility and thereby increasing the likelihood of its elimination through
the kidney. In addition, this process provides for the possibility of
attachment to hydroxyl groups of other polar moieties such as sulfate,
glucuronic acid, and glutathione, which could further facilitate excretion
of the chemical via the kidney or biliary active transport systems.
In addition to facilitating excretion of the foreign chemical, the
monooxygenase activity may also create a more toxic chemical. For example,
the action of cytochrome(s) P-450 converts the pesticide parathion to the
toxic form, paraoxon, and converts the polycyclic aromatic hydrocarbon
carcinogens to the proximate carcinogens. Several chemicals, such as
phenobarbital and 3-methylcholanthrene, increase (induce) the amount of
cytochrome(s) P-450 and related enzymic activities following -in vivo
administration. The various inducers do not increase all enzymic activities
equally, nor are the increases in enzymic activity necessarily proportional
to the increase in total cytochrome(s) P-450. Several chemicals, such as
piperonyl butoxide, inhibit the enzymic activity of cytochrome(s) P-450 when
they are administered in vivo or when they are added to the microsomes in
vitro.
Previous Studies of Xenobiotic Compounds in Fish
The biotransformation of drugs and xenobiotic substances in fish has
only recently received attention, although much progress has been made in
this area in mammals. Early studies concerned with biotransformation of
drugs in several species of fish led to the conclusions that fish do not
metabolize drugs to any extent and that they do not conjugate foreign
phenols (Brodie and Maickel 1962).
The lack of need for these biotransformation processes was supported
teleologically by the aqueous environment of fish and by their ability to
excrete lipid soluble drugs through the gills into the sink of that
-------
environment. The implication of this concept is that an outward
concentration gradient always exists for this important excretory process
when compounds are injected into fish and provisions are made to maintain an
adequate outward concentration gradient. The work of Maren et al. (1968)
has provided evidence that lipid solubility is an important determinant of
the speed of outward gill diffusion and that injected compounds which are
ionized at the pH of fish blood have longer half-lives than those which are
unionized. While such diffusion is important in situations involving
ingested foreign compounds, a common and potentially harmful situation
occurs when fish are exposed to a constant concentration of a given compound
in the aqueous environment. In such an instance the gradient is inwards,
and, even after the steady state has been established, no net outward
gradient exists across the gill. Any protection against the offending
substance must therefore be afforded by biotransformation and excretion
mechanisms when and if they exist.
Several reports have shown that both freshwater and saltwater fish have
the enzymes to oxidize many drugs and foreign compounds (Adamson 1967,
Dewaide and Henderson 1968, Buhler and Rasmussen 1968) and that these
enzymes appear to be microsomal in nature and require NADPH and 0~• The
levels of these enzymes in both fish liver and kidney are species dependent
but, in general, seem to be about 5-10 times lower than those found in
mammals (Adamson 1967). Nitroreductase, glucuronyl transferase, and
sulfotransferase have also been found in many species of fish (Button and
Montgomery 1968, Adamson et al. 1965). In vitro metabolism of parathion,
guthion, diazinon, and dimethylnitrosamine by fish liver has been
demonstrated in several laboratories (Potter and O'Brien 1964, Murphy 1966,
Hogan and Knowles 1972, Montesano et al. 1973). Dewaide (1971) has reviewed
a wide range of studies concerning the properties of drug-metabolizing
enzymes in fish. More recently there have been reports of mixed function
oxidase activity in many extrahepatic tissues in Raja erinecea (Bend et al.
1973) and Stenotomus ber>sieolor> (Stegeman et al. in press). The great gap
in the knowledge of the disposition of foreign compounds in fish is caused
by the lack of in vivo studies that are needed to assess the functional
significance of the in vitro work that has demonstrated the presence of
these enzymes.
Several reports document the occurrence of biotransformation reactions
in fish in vivo. Huang and Collins (1962), in a study of the disposition of
injected p-nitrobenzoic acid in flounder, dogfish, and goosefish,
demonstrated the presence of several polar conjugates of this compound in
the urine collected during the experiment, even though an adequate
concentration gradient was maintained for outward gill diffusion. These
conjugates were thought to be derivatives of glycine, glucuronic acid, and
acetic acid. Lotlikar et al. (1967) reported that 2-acetoaminofluorene is
hydroxylated by rainbow trout in vivo, and Hunn et al. (1968) have noted the
presence of the N-acetyl-derivative of the fish anesthetic,
tricainemethanesulfonate, in blood of trout exposed to this agent.
Gutenmann and Lisk (1965) noted the in vivo conversion of 4-(2,4-
dichlorophenoxybutyric acid [4-(2,4-DB)J to 2,4-dichlorophenoxyacetic acid
(2,4-D) by bluegills. In an evaluation of endosulfan (Thiodan) as a fish-
control agent, Schoettger (1970) reported the occurrence of a conjugated
8
-------
metabolite in the bile of white suckers, but this compound was not
characterized. Rotenone, an insecticide and fish-control agent, can
apparently be metabolized by carp in vivo (Fukarai et al. 1969). In a study
of the dynamics of 2,4-D in channel catfish and bluegills, Shultz (1973)
reported the occurrence of as many as six metabolites of this compound in
fish muscle.
A study of 14C-2,4-D in bluegills (Macek et al. 1975) showed not only
that metabolism is apparently important in the elimination of some chemicals
from fish but also that some chemicals may induce systems responsible for
their own metabolism. Some of the chemicals studied by this group were
accumulated by bluegills so that tissue levels increased for 1-2 weeks or
even longer. Others, however, showed this accumulation for only a few days
followed by an almost complete loss of tissue ^C despite continued
exposure, suggesting that some process responsible for rapid elimination may
have been initiated.
In vivo studies using marine fish have indicated that the species
studied were able to hydroxylate several components of petroleum oil and
that the bile was a major storage site of the polar metabolites (Lee et al.
1972). The information gained is of great significance since the
elimination of these highly lipid soluble compounds from the species studied
had a component which was dependent upon biotransformation rather than
solely outward diffusion of the unchanged chemical. Another report
indicated that rainbow trout can hydroxylate 3,4-benzpyrene, a carcinogenic
constituent of crude oil (Pederson et al. 1974).
One of the most interesting problems in this area concerns the effects
of inducers on microsomal enzyme activity in various species of fish. One
of the earliest studies with DDT (Buhler 1966) indicated that several
hepatic microsomal enzymes could be induced in trout, but subsequent reports
demonstrated great variability in response to both DDT and phenylbutazone
(Bend et al. 1973, Addison et al. 1977). Dewaide (1971) reviewed a wide
range of work concerning the properties of microsomal enzymes in fish and
suggested that the levels of activity and response of microsomal enzymes to
inducers could be important in determining the susceptibility of fish to
toxic substances in the environment. The application of concepts gleaned
from studies in mammals demonstrates that inducers of the 3-
methylcholanthrene (3-MC) type can induce arylhydrocarbon hydroxylase (AHH)
activity in several marine species (Payne and Penrose 1975, Bend et al.
1973). In fact, the level of AHH activity in trout has been closely
correlated with the level of oil pollution in some bodies of water (Payne
1975).
Goals of the Research
For the present project we selected for investigation several questions
whose answers will increase knowledge of the ability of fish to cause the
biotransformation of xenobiotics. Studies of these biotransformations in
vitro have focused on the liver because of its central role in such
reactions in the intact fish. Published work on this subject has utilized
homogenization and fractionation of liver by the same methods used for
-------
mammalian liver without characterizing the fractions obtained from fish
liver. We will therefore begin with the characterization of the subcellular
fractions of rainbow trout liver and proceed with validation of various
enzyme assays in the appropriate fraction(s). These assays are utilized for
characterization of xenobiotic metabolizing activity. Subsequently, the
xenobiotic metabolizing ability [microsomal monooxygenase, cytochrome(s) P-
450] of the livers of normal and induced trout will be characterized.
Biotransformation also will be evaluated -in vivo by characterization of
the metabolites found in bile and tissues of the organic pollutants listed
earlier after exposure of fish to the C-labeled chemicals. The effect of
inducers and inhibitors on the production of such metabolites also will be
examined.
ACCUMULATION AND ELIMINATION OF XENOBIOTICS BY FISH
The accumulation of water-borne chemicals in fish is dependent on a
variety of processes. Although many studies have been carried out on the
uptake and bioaccumulation of organic chemicals in fish, little has been
done in studying a series of related chemicals in order to correlate their
structural features with the processes which govern the biological
disposition of foreign chemicals. Fromm and Hunter (1969) reported that
dieldrin was transferred across perfused rainbow trout gills when blood or
blood plasma was used as the perfusing solution but not when a non-lipid
solution was used. They suggested that the dieldrin was more soluble in
blood (containing lipoprotein) than in the exposure water and therefore
crossed the gill barrier into blood, thereby entering tissues where even
greater solubility of dieldrin in tissue lipids exists. Hamelink et al.
(1971) expanded this idea and suggested that in general organochlorine
compounds of low water solubility are accumulated on the basis of exchanges
dependent on increasing lipid character.
More recently, Neely et al. (1974) related bioaccumulation of a given
compound to its octanol^water partition using the well-known Hansch
coefficient utilized by the pharmaceutical industry. These workers reported
a linear relationship between the log of the partition coefficient and the
log of the biocencentration factor for many compounds. This method,
however, and any other method which predicts bioaccumulation solely on the
water solubility of the compound, ignores the possibility of metabolism of
the compound in fish. These investigators also have proposed an accelerated
method for predicting bioconcentration by use of the rate constants for
uptake and washout of a chemical by fish (Branson et al. 1975). Their
method, however, treats dilution by growth as elimination and may therefore
be misleading.
Relatively little information is available in the reviewed literature
on the uptake and disposition of coal constituents by freshwater fish.
Apparently, the most water soluble (water-extractable) constituents of coal
and petroleum are similar. Among the 20 chemicals found in an aqueous coal
leachate were naphthalene, two methylnaphthalenes, and two
dimethylnaphthalenes (Carlson and Caple 1979). Naphthalene and
methylnaphthalenes are also among the substituents of petroleum and
10
-------
petroleum products which are most readily extracted into water (Boylan and
Tripp 1971, Anderson and Neff 1975, Larson and Weston 1976). Furthermore,
naphthalene and methylnaphthalenes are the organics appearing in the Gulf
Killifish (Fundulus similus) at the highest levels following exposure to the
water-soluble fraction of No. 2 fuel oil (Neff et al. 1976). The several
reported studies of the uptake of naphthalene and related chemicals by
marine species of fish are therefore of interest. Lee et al. (1972) studied
the uptake, metabolism, disposition, and elimination of ^C-naphthalene
and ^H-3, 4-benzopyrene by mudsucker (Gillichtye mirdbil-i), tidepool
sculpins (Oligocottue maeulosus), and sand dab (Citharichthys etigmaeue).
During exposure to either of these chemicals, fish of all three species
accumulated C or H to levels very much higher than the aqueous exposure
level during short term exposure (2 h or less). When the fish were placed
in fresh seawater following exposure to the labeled chemical, the C
accumulated from C-naphthalene was released very rapidly, while the H
from H-3,4-benzopyrene was released much more slowly. Gall bladder bile,
urine, and tissue extracts from these fish were examined for the presence of
biotransformation products by thin-layer chromatography. The radioactive
peaks were tentatively identified by comparison to published Rj values. For
both chemicals the major product appeared to be a dihydrodiol.
In order to predict accurately the bioaccumulation of a chemical in
fish the ability to incorporate a metabolism factor into the expression of
the phenomena of uptake and elimination by the whole organism is
important. The ability of rainbow trout to metabolize the lampricide TFM by
formation of TFM-glucuronide is so important that it apparently explains the
difference in toxicity of TFM to rainbow trout and to lampreys, which cannot
form TFM-glucuronide (Lech and Statham 1975).
In this study we will examine the uptake and elimination of the
chemicals listed under organic pollutants. In addition the effects of
inducers or inhibitors of biotransformation on tissue levels, on rates or
elimination, and on the form of the pollutant present will be examined. In
particular, we will attempt to evaluate the role of metabolism in the
accumulation and elimination of these chemicals.
Previous studies in this laboratory have shown that certain foreign
chemical and/or their metabolites appear in fish bile at concentrations many
times higher than the concentrations in the water (Statham et al. 1976). We
have suggested that bile sampling might therefore serve as a monitoring
method for selected environmental polutants. With this in mind, the biliary
forms of the various chemicals under study will be characterized.
METHODOLOGY
Fish
Rainbow trout (Salmo gairdneri) were obtained from the Kettle Moraine
Springs Trout Hatchery, Adell, Wisconsin, and bluegill sunfish (Lepomis
macroahirus) and carp (Cyprinus earpio) were supplied by the U.S. Department
of the Interior, Fish and Wildlife Service, Lake Mills National Fish
Hatchery, Lake Mills, Wisconsin, and Fish Control Laboratory, LaCrosse,
11
-------
Wisconsin. These fish were maintained in flowing charcoal-filtered tap
water at 12°C (the temperature at which the experiments were performed) for
at least 1 week prior to use and were fed commercial trout pellets 3
times/week. A cycle of 12 h light, 12 h dark was used. Exposures of
saltwater sheepshead (Archosargue probatoeephalus) were performed at the
National Institutes of Environmental Health Sciences Laboratory at
Marineland, Florida, at 15°C on the day following capture.
Pretreatment of Fish for Studies of Induction of Hepatic Xenobiotic
Metabolizing Activity
3-Naphthoflavone, 3-methylcholanthrene, 2,3-benzanthracene, Aroclor
1254, Aroclor 1242, and Firemaster BP6 were administered to trout by
intraperitoneal injection as solutions in corn oil (1 ml/kg). Doses of
these compounds varied from 0 to 275 mg/kg. Fhenobarbital (65 mg/kg) was
administered intraperitoneally as an aqueous solution (1 ml/kg). Control
fish received either corn oil or water alone. Careful injection resulted in
no observable leakage of compounds from the injection site. After injection
fish were kept in 50 liter tanks (5-7 fish/tank) until sacrifice.
Exposures of Fish to C-labeled Chemicals
Static Exposures—
In order to study the uptake, elimination and metabolism of the C-
labeled chemicals by fish, some experiments were conducted using relatively
short exposure times. When such experiments required exposure to the C-
labeled chemical for 36 h or less, the fish were exposed to the chemical in
a non-flowing aqueous system. When the exposures were performed for the
purpose of studying biotransformation, the fish were sacrificed at the end
of the exposure period and treated as described in later sections on Tissue
Treatment and Metabolite Studies. When exposures were performed for the
purpose of studying uptake and elimination, fish were sacrificed at various
times during the exposure period and during a subsequent elimination period
in fresh-flowing water and treated as described under Tissue Treatment.
Continuous Flow Exposures—
For uptake and elimination studies, rainbow trout (3-6 g), carp
(2-6 g), or bluegill sunfish (0.7-1.4 g) were maintained in aqueous C-
labeled naphthalene, 2-methylnaphthalene, or 1,2,4-trichlorobenzene for 4-5
weeks. The concentration of these chemicals in the exposure water averaged
0.013 to 0.023 mg/liter, and the individual values are included with the
results. The continuous-flow delivery system was similar to the system of
Mount and Brungs (1967) as modified by DeFoe (1975). A solution of the 14C-
labeled chemical in acetone was added to the water at 3 pliter/liter. The
water flow was approximately 8 liters/h. Because of the high volatility of
the chemicals being studied, the water was aerated in the distributing
system before the C-labeled chemical was added. The exposure was
initiated by the addition of the fish to 95 liters of water containing the
appropriate chemical. Groups of five fish were removed at intervals for the
determination of tissue levels of C. After approximately 4 weeks of
12
-------
exposure to the ^C-labeled chemical, the remaining fish were transferred to
fresh-flowing water to monitor the elimination of that chemical. Again,
groups of five fish were removed at suitable intervals to determine
tissue -^C levels. Water levels of each chemical were monitored by counting
the -^C present in aliquots of the water during the exposure and elimination
periods. The fish were fed twice per week, and sampling was usually done at
least 2 days after feeding.
Effect of Induction of Liver Xenobiotic Metabolizing Enzymes on Disposition
and Metabolism of Chemicals i,n
Rainbow trout (80-100 g) were treated with corn oil or 2,3-
benzanthracene in corn oil as described earlier. Both groups were placed in
tanks for 48 h after which the trout were exposed to C-2-methylnaphthalene
(0.05 mg/liter) in 50-liter tanks for 6 h. The fish were returned to fresh-
flowing water and sampled at various intervals for the determination of C-
labeled material in blood, muscle, liver, and bile.
Groups of eight rainbow trout (70-120 g) were treated with corn oil or
a solution of BNF in corn oil as described above. After 48 h the groups of
control fish (corn-oil injected) and induced fish (BNF-injected) were placed
in tanks containing C-labeled naphthalene, 2-methylnaphthalene, or 1,2,4-
trichlorobenzene in 50 liters of water. The fish were sacrificed after 24 h
exposure to the C-labeled chemical (72 h after the injections). Samples
of bile, blood, liver, and muscle were taken. These samples were used
for C measurements, for determining the percentage of C present as
metabolites (polar material), or both.
Studies also were conducted on the effects of the microsomal inhibitor
piperonyl butoxide on the biotransformation of pentachloroanisole and di-2-
ethylhexylphthalate by rainbow trout •In vivo. In these experiments the
trout were exposed to 1 mg/liter piperonyl butoxide for 24 h, followed by
coexposure to piperonyl butoxide plus the C-labeled chemical for 24 h.
Preparation of Subcellular Fractions of Liver
The procedure for the preparation of subcellular fractions of fish
liver used in the initial studies is summarized in Figure 1. Fish were
sacrificed by cervical dislocation. The livers were removed, minced, and
subjected to the homogenization and fractionation procedures listed in
Figure 1. A 1 g sample of fish liver was assumed to have a volume of 1 ml
and the livers were homogenized in four volumes of 0.25 M sucrose. All
operations were carried out at 4°C. Each particulate fraction was suspended
in 0.25 M sucrose in a volume equal to 16.7% of the starting homogenate and
was repelleted at the same gravitational (g) force. The second supernatant
was combined with the original supernatant obtained for the respective
fraction and subjected to the next higher g force to obtain the subsequent
fraction. All particulate fractions were suspended finally in a volume of
0.25 M sucrose equal to that of the starting homogenate. To give a more
homogenous suspension, all membranous fractions (including starting
homogenate fractions) were sonified for 3 x 10 sec periods separated by 30
sec periods with a Branson Sonifier Cell Disrupter Model W140 D (20,000 Hz,
13
-------
s
CM
•
O
*-[._
o
tf>
E
"o
>
^
J
_c
-Q
d)
'c
g"
E
o
-o
re
T3
o
c
'i
£
(/>
o5
>
_Ii
to
0)
O
to
sO
s^
o3
N
1
01
o
0
V)
"o>
1
g
"a5
CD
o
^
*-*
0
E
re
nC
'I
o>
0
o
/A
E c
O "-1
fe °
•*- >
> TO
re 01
01 *j O
l^J tO —
0 "re
^ E oo
"re n. ±i
^5 *^
1 . M I
•^
re
"o
3
c
E
o
E
O
o
0)
4J
3
O
c
•H
CS
M
M-l
O
C
O
•rl
u
C8
U-t
!M
O
O
CO
0)
M
3
M
•H
-------
400 watts), amplitude setting of 6, fitted with a microtip. Between each
sonification period, a 30 sec period of cooling at 4°C was introduced to
prevent temperature increases.
A slightly different protocol was used when the only subcellular
fraction required was the microsomal fraction. In this case the livers were
minced and washed 3 times with ice-cold 0.154 M KC1 to remove adhering
hemoglobin. The minced livers were homogenized for six complete strokes in
four volumes of 0.25 M sucrose using a motor-driven Potter-Elvejhem glass-
teflon homogenizer. The homogenate was centrifuged at 8,500 x g (r =8.3
cm) for 20 min using a Sorvall-type-24 rotor and RC-5 centrifuge. The
resulting supernatant was centrifuged at 165,000 g (r =5.7 cm) for 60 min
using a Beckman type 65 rotor and Model L5-65 ultra-centrifuge. The
microsomal pellet obtained was resuspended in 0.154M KC1 and the microsomes
residimented at 165,000 x g for 60 min. The washed microsomal pellet was
resuspended in 0.25 M sucrose to give a final concentration equivalent to
1 g wet weight liver/ml. All operations were performed at 0-4°C, and the
microsomes were utilized on the day of preparation.
A different protocol also was used to prepare trout liver subcellular
fractions for studying metabolism of C-di-2-ethylhexylphthalate so that
results could be compared to those reported for another species (Stalling et
al. 1973). The livers were quickly removed and placed in ice-cold 0.154Af
KC1. The livers were weighed, minced, and rinsed with ice-cold 0.154M
KC1. A 20% homogenate of the livers in 0.154M KC1 was-then made by five
up-and-down cycles of a motor-driven homogenizer of the Potter-Elvejhem type
having a glass vessel and a Teflon pestle.
Subcellular fractions were prepared by differential centrifugation at
4°C. The liver homogenate was first centrifuged for 10 min at 2,000 g in a
Sorvall model RC-5 refrigerated centrifuge. The pellet from this
centrifugation generally was not utilized, but the supernatant fraction was
used as prepared or was further fractionated in a Beckman model L-2
ultracentrifuge. The pellet resulting from a 20-min centrifugation of the
2,000 g supernatant fraction at 10,000 g was resuspended with use of the
tissue homogenizer in a volume of 0.154 M KC1 equal to or less than the
volume of the supernatant fluid removed. This resuspended fraction was
referred to as the mitochondrial fraction. The 10,000 x g supernatant
fraction was centrifuged at 100,000 x g for 1 h. The 100,000 x g pellet was
resuspended in 0.154 M KC1 as described for the 10,000 x g pellet, and was
designated the microsomal fraction.
In studies in which serum was used, blood was obtained from the caudal
vein of rainbow trout and allowed to clot. The remaining liquid was
centrifuged at 1,500 x g for 10 min, and the supernatant fluid was diluted
5- or 10-fold with 10 m¥ phosphate buffer, pH 7.2.
Enzyme Assays and Spectral Measurements
Succinic dehydrogenase activity was measured using a reaction mixture
containing 10 mV phosphate (pH 7.4), 10 mg bovine plasma albumin, 1 wM KCN,
and 1 mg horse heart cytochrome C (Green et al. 1955) in a final volume of
0.9 ml. After recording the baseline at room temperature, the reaction was
15
-------
initiated by adding 0.1 ml of 50 m¥ sodium succinate, and the change in
absorbance was measured at 550 nm with a 1 cm path in a spectrophotometer
(Gilford) with a recorder.
Acid phosphatase was assayed using p-nitrophenyl phosphate as substrate
(Gianetto and deDuve 1955). The g-glucuronidase activity was measured with
phenolphthalein glucuronide as substrate (Gianetto and deDuve 1955). Acid
phosphatase, 3-glucuronidase and all enzyme assays described below were
carried out at 25°C. This temperature is considered optimal for enzymatic
activities of fish liver (Dewaide 1971) rather than 37°C, which is a usual
temperature for assaying many enzymatic activities of mammalian
preparations. The acid phosphatase and 6-glucuronidase assays were stopped,
in the first case by the addition of 1.0 ml of 2.0 M glycine (pH 10.7) and
in the second by the addition of 1.5 ml of 0.13 M glycine, 60 mM NaCl, and
80 vM NaoCOn (pH 10.7). The reaction tubes were centrifuged at
approximately 30,000 x g for 10 min before absorbance was read at 420 nm.
Cathepsin D activity (Gianetto and deDuve 1955) was assayed using a
final concentration of 0.5% hemoglobin as substrate. After the reaction was
stopped with 1 ml of 5% trichloracetic acid (TCA), the samples were
centrifuged at approximately 30,000 x g for 10 min and the adsorbance read
at 280 nm.
Alkaine phosphatase was assayed by incubating the enzyme preparation in
a final volume of 0.5 ml containing 0.1 M ethanolamine (pH 9.5), 1.5 vM
sodium floride, and 5 mV p-nitrophenyl phosphate at 25°C for 30 min. After
the reaction was terminated with 1 ml of 2 M glycine (pH 10.7), the samples
were centrifuged at approximately 30,000 x g for 10 min before the
adsorbance was read at 420 nm.
Lactic dehydrogenase activity was assayed with a reaction mixture
containing 50 mM TES [sodium N-tris (hydroxylmethyl) methyl-2-aminoethane
sulfonic acid], 1 mW dithiothreitol, and 0.2 mM NADH. After the baseline
was recorded, the reaction was initiated with 0.1 ml of 0.1 M pyruvate and
the change in adsorbance was measured at 340 nm.
Uridine diphosphoglucuronic acid (UDPGA)-glucuronyl transferase
(indicated subsequently as glucuronyl transferase) was assayed at 25°C with
a reaction mixture containing 100 mW sodium phosphate (pH 7.0), 0.2 mM
MgCl2, 5 riM saccharo-l,4-lactone, 0.5 mV UDPGA, and 0. 5 m¥ 14C-3-
trifluoromethyl-4-nitrophenol containing approximately 3.0 x 15 counts/min
(CPM) in a final volume of 1.0 ml. The reaction was stopped after 10 min
with 0.2 ml of 10% TCA, which was followed by the addition of 1.0 ml water
to each tube. Extraction of unreacted, labeled substrate was done with 4 ml
of benzene :ether (1:1 v:v) using a 10 min shaking time with a mechanical
shaker after which the upper phase was removed by aspiration. This
procedure was repeated twice. An aliquot (0.5 ml) of the lower aqueous
phase was counted in 15 ml of ACS scintillation cocktail (Amersham/Searle).
A reaction tube without added UDPGA was assayed for each cellular function,
and the radioactivity found under these assay conditions was subtracted from
that found in the presence of UDPGA.
16
-------
The 5'-nucleotidase assay was carried out at 25°C with a reaction
mixture containing 50 mV TES (pH 7.5), 1 mM ethylendiamine tetracetic acid
(EDTA), 5 nW MgCl2, and 0.1 mV 3H 5'-adenosine monophosphate (AMP)
containing approximately 50,000 CIM in a final volume of 250 yliter. After
the incubation period (20 min), the reaction was terminated by boiling for
1 min. The 3H-adenosine was separated from the labeled substrate on a small
4.5 cm column of DEAE-Sephadex A-25, contained within a Pasteur pipette. A
similar "pencil" column technique has been described for the separation of
labeled adenosine from cyclic AMP in the assay of cyclic nucleotide
phosphodiesterase (Huang and Kemp 1971). The 3H 5'-AMP, eluted with 3.0 ml
50 mV Tris HC1 (pH 7.5), was counted in a Packard liquid scintillation
spectrometer (Model 3310) with 10 ml of a premixed scintillation cocktail
(ScintiVerse, Fisher Scientific Company). An aliquot of the assay mixture
containing 3H 5'-AMP was counted in 3.0 ml 50 mW Tris HC1 buffer under the
same conditions as the assay samples and no quench correction was required.
Rotenone-insensitive NADH or NADPH-cytochrome C reductase was assayed
in the presence of 1.5 yAf rotenone and the reduction of cytochrome C at 550
nm was followed (Sottocasa et al. 1967). Rotenone for the enzyme assay was
prepared by the drop-wise addition of 200 yliter of 0.225 M rotenone in
dioxane in 10 ml of 10% albumin; 0.1 ml of this mixture was used in the
assay to give a final rotenone concentration of 1.5 yAf.
Glucose-6-phosphatase activity was assayed at pH 6.0 by measuring the
inorganic phosphate released from glucose-6-phosphate in a reaction mixture
containing KF and EDTA to minimize interference in. the assay by acid and
alkaline phosphatase activities (Hubscher and West 1965). After the
reaction was stopped with 10% TCA, the samples were centrifuged at
approximately 30,000 x g for 10 min, and an aliquot of the clear supernatant
was assayed for inorganic phosphate with ascorbic acid and ammonium
molybdate (Ames 1966).
The benzopyrene hydroxylase assay, used in the present study, was based
on the assay initially described by Hansen and Fouts (1972). The reaction
mixture contained 0.1 M Tris-HCL (pH 7.4), 5 mW glucose-6-phosphate, 2 units
glucose-6-phosphate dehydrogenase, 1 mM NADP, and 5 yAf ^C-benzo[a]pyrene
containing approximately 10 CPM and enzyme in a final volume of 2.5 ml.
The reaction stopped after 5 min with 1.0 ml of cold acetone and placed in
an ice bath (4°C). Hexane (5.0 ml) was added to each sample and the mixture
was shaken for 20 min. Then 2 N NaOH (2.5 ml) was added to each tube, and
the shaking was continued for an additional 20 min. The samples were
centrifuged at 2,500 rpm with an IEC centrifuge for 10 min, the upper
(hexane) layer was removed by aspiration, and a 1.0 ml aliquot of the
aqueous layer was counted in a liquid scintillation spectrometer with 15 ml
ACS scintillation cocktail in the presence of 100 yliter of glacial acetic
acid to minimize chemiluminescense.
Ethylmorphine-ff-'demethylation was determined by measurement of
liberated HCHO by a modified method of Anders and Mannering (1966).
7-Ethoxycoumarin-Cl-deethylation was measured by the direct fluorimetric
procedure of Ullrich and Weber (1972). Final quantities of reactants in the
17
-------
cuvette were 100 nmole of NADPH, 3.2 mole of glucose-6-phosphate, 2 units
of glucose-6-phosphate dehydrogenase, 5-450 nmole of ethoxycoumarin,
0.1-0.3 mg of microsomal protein, and 66 roV Tris-HCl buffer (pH 7.4) in a
final volume of 1 ml.
7-Ethoxyresorufin-<9^deethylation was determined by the method of Burke
and Mayer (1974). Final quantities of reactants in the cuvette were 500
nmol NADPH, 0.1-0.5 mg microsomal protein, 15-600 pmol ethoxyresorufin, and
66 mW Tris-HCl buffer (pH 7.4) to 1 ml total volume.
Temperature optima for these monooxygenase assays were 25°C for
arylhydrocarbon hydroxylase and ethylmorphine-^-demethylase and 29-30°C for
ethoxycoumarin- and ethoxyresorufin-<9-deethylases.
Protein was determined by the method of Ross and Schatz (1973) using
crystalline bovine plasma albumin as the standard.
Spectral Measurements—
All measurements were made using either an Aminco DW2 UV/VIS
Spectrophotometer or a Gary 219 Spectrophotometer. The \ for the CO
complex of Na2So04-reduced hemoprotein(s) P-450 were determined at a protein
concentration of 1-2 mg/ml. The Spectrophotometer was calibrated using a
holmium oxide filter before and after use. An extinction coefficient of
100 m¥ cm for the difference in absorbance between 450 and 510 run of the
carboxyferrocytochrome P-450 minus ferricytochrome P-450 difference spectrum
was utilized (Estabrook et al. 1972). This method involves reducing the
contents of the sample cuvette with ^28264 and gassing both cuvettes with
CO, hence eliminating spectral interference due to haemoglobin.
Type I and type II oxidized binding spectra of various compounds with
the trout hepatic microsomes were obtained as described by Schenkman et al.
(1967). The substrates were dissolved in N,#-dimethylformamide and added as
Viliter quantities to the sample cuvettes. Equivalent quantities of
dimethylformamide were added to the reference cuvette. Dimethylformamide did
not elicit any binding spectra of its own at the concentrations employed in
this study.
Ethylisocyanide-ferrocytochrome P-450 difference spectra were obtained
at pH 7.4 as described by Imai and Sato (1966).
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
was performed at room temperature using a modified system of Laemmli and
Favre (1973) as described by Dent et al. (1978). A 15-cm-long 7.5%
acrylamide resolving gel was utilized. Staining for protein was with
Coomassie brilliant blue R-250 and for peroxidase activity the precedure of
Thomas et al. (1976) was used.
DEHP Metabolism in Vitro—
The in vitro metabolism of DEHP was assessed by incubation of the
various tissue fractions with ^C-DEHP and determination of the amount of
18
-------
DEHP remaining and of metabolites formed during the incubation. The
incubation solution consisted of 0.4 ml of tissue preparation in 0.154M
KC1, 0.4 ml of 10 irff phosphate (pH 7.2), 0.1 ml of 10 nW MgCl2, and either
0.1 ml of 20 nM NADPH (pH 7.2) or 0.1 ml of water/ml of incubation medium.
Total volume was 1.0 or 2.0 ml. The addition of compounds of low water-
solubility, DEHP, 2,4-DBE, and piperonyl butoxide (PBO), was accomplished by
the addition of small volumes (5-40 y liter) of solutions of these materials
in methanol, which was evaporated off before addition of the aqueous
components. 14C-DEHP was present at 5 VM, 14C-2,4-DBE at 10 u#, and PBO at
2 nW. A slightly different method for the addition of PBO was utilized
because of its low solubility in water. A premix of the ionic constituents
of the incubation medium was shaken with PBO followed by centrifugation and
decanting of the aqueous layer. This premix with PBO was added to 14C-
DEHP. The concentration of PBO in the premix was determind (Bhavnagary and
Ahmed 1973) and found to result in a final concentration of 9 x 10"^ M PBO.
Incubations were initiated by the addition of the appropriate tissue
fraction to a chilled tube containing the other required materials which was
placed in a Dubnoff Metabolic Shaker at 22°C. Incubations were terminated
by the addition of 0.2 ml of 10 N HC1. Extraction of the acidified
incubation medium three times with 1.0 ml of diethyl ether removed over 98%
of the radioactivity. The pooled ether extracts from each tube were reduced
in volume to approximately 0.3 ml to facilitate further analysis. The
extent and nature of the metabolism occurring during these incubations were
evaluated by thin layer chromatography (TLC) of the extracts on plates
coated with silica gel containing a UV indicator which served to locate the
phthalate standards which were run along with the incubation extracts. The
solvent systems used were CHCl-j:MeOH:HOAc in the ratios 143:7:2 and 5:1:1
(v:v:v) for the DEHP studies, and hexane: ace tone (1:1) for 2,4-DBE. The
developed plates were monitored using a Packard Model 7201 Radiochromatogram
Scanner to locate the radioactive peaks which were quantified by scraping
gel segments and counting them in a scintillation counting system (Searle
Analytic Company Model 300). The percentage of total ^4C present in each
metabolite peak was calculated and converted to nanomoles based on initial
nanomoles of DEHP and 2,4-DBE.
Tissue Treatment for C Determinations
Aliquots of bile and blood were added to 15 ml of ACS scintillation
mixture for 14C counting. Similarly, aliquots (1 or 2 ml) of the various
exposure solutions were monitored for C; all exposure levels of C-
labeled chemicals specified in the results are based on these measurements.
Weighed portions of solid samples were dissolved in NCS tissue solubilizer
at 48°C for 24 h before addition of the ACS scintillation mixture. On some
occasions entire small fish or carcasses were dissolved and aliquots of the
resulting solution were used for C determinations.
After addition of the scintillation mixture each vial received
40 y liter of glacial acetic acid followed by 24 h of heating at 48°C and
24 h of cooling before counting to reduce chemiluminescence. Radioactivity
counting was done with a Searle Analytic Company Isocap/300 Liquid
Scintillation System. A computer program was used to convert the
19
-------
radioactivity data to the amount of parent C-labeled chemical plus
metabolites present in each tissue expressed as yg/g.
Metabolite Studies
Bile was examined for the presence of metabolites of the C-labeled
chemicals by TLC or by solvent partitioning. After TLC, 1-cm segments of the
silica gel from origin to solvent front were scraped and subjected to
radioactivity counting. The locations of the radioactivity were compared to
those of appropriate standards. Aliquots of bile were partitioned between 2
ml of hexane and 2 ml of dilute phosphate or 2-amino-2-(hydroxymethyl)-l,4-
propanediol buffers (pH 7.4 or 11.0) followed by measurement of the
radioactivity present in each phase. After partitioning at pH 7.4 only
conjugated or acidic metabolites appeared in the aqueous phase, while at pH
11 the phenolic metabolites also appeared in the aqueous phase.
Muscle and liver tissues were placed in cold acetone at the time of
dissection. They were homogenized using a Waring blender in a chilled
homogenization vessel. The tissues were extracted at least twice with
acetone, once or twice with hexane, and usually at least once with ethanol
or methanol. In the case of muscle this final alcohol extraction included
an additional homogenization. Extractions of radioactivity were at least 95%
complete as evaluated by digestion and radioactivity measurements on
aliquots of the extraction residues. Aliquots of these extracts were
examined by TLC and hexane^water partitioning or both to evaluate the
presence of metabolites.
To assist in the identification of possible glucuronide conjugates of
biotransformation products, hydrolysis based on catalysis by 3-glucuronidase
was utilized.
Aliquots of solutions of the metabolites were evaporated to dryness and
the residue was dissolved in 0.05 M phosphate buffer (pH 6.8).
g-glucuronidase solution (1 Sigma unit/yliter) was added to the metabolite
solution to give a final concentration of 200 units/ml, and the final
solution was incubated at room temperature. In some cases, duplicate
aliquots were utilized to check whether saccharo-l,4-lactone, a specific
inhibitor of g-glucuronidase (Levvy 1952), inhibited the hydrolysis. When
the hydrolysis was done for analytical purposes, an aliquot of the
incubation solution was applied directly to a TLC plate. When the
hydrolysis was done for preparative purposes, the incubation solution was
extracted several times with diethyl ether after acidification. In this
report the term "hydrolyzed metabolite" refers to the results of in vitro
incubation with g-glucuronidase rather than to any in vivo metabolic
reaction.
Thin-Layer Chromatography
Aliquots of bile and of various tissue extracts were subjected to TLC
on silica gel plates using various solvents, depending upon the compounds of
interest. The various samples were co-chromatographed with appropriate
standards. After chromatography the standards were located by use of a
20
-------
hand-held shortwave UV light while the radioactivity was located by scraping
a 1 cm segment of the silica gel from origin to solvent front and
scintillation counting of the individual segments.
Materials
NADP+, NADPH, NADH, glucose-6-phosphate, glucose-6-phosphate
dehydrogenase, 7-hydroxycoumarin (umbelliferone), saccharo-l,4-lactone,
glucuronidase (bacterial type II), phenobarbital, and UDPGA were obtained
from Sigma Chemical Company (St. Louis, Missouri).
7-Ethoxycoumarin was synthesized by the method of Ullrich and Weber
(1972) and 7-ethoxyresorufin (7-ethoxyphenoxazone) was synthesized by Dr. S.
R. Challend (Wellcome Research Labs, Beckenham, Kent, U.K.) from resorufin
(7-hydroxyphenoxazone, Eastman Organic Chemicals, Rochester, New York).
Aroclors 1254 and 1242 were generous gifts from Monsanto Chemical
Company while metyrapone (Metopirone) was kindly donated by the Ciba-Geigy
Corporation (Sumitt, New Jersey). Dr. D. Rickert (Chemical Industry
Institute of Toxicology, Raleigh, North Carolina) generously donated
Firemaster BP6.
g-Naphthoflavone (5,6-benzoflavone), and a-naphthoflavone were
purchased from Aldrich Chemical Company (Milwaukee, Wisconsin).
5'-AMP was obtained from PL Biochemicals, Milwaukee, Wisconsin, and
Fraction V bovine plasma albumin from Rheis Chemical Company, Kankakee,
Illinois.
Ethyl isocyanide was kindly donated by Dr. R. Philpot (National
Institute of Environmental Health Sciences, North Carolina).
All other chemical reagents and solvents used in the enzyme assays were
of the highest commercial quality available.
8-14C-2-Methylnaphthalene (specific activity, 8.0 mCi/mmol) was
supplied by California Bionuclear Corp. (Sun Valley, California).
1-14C-Naphthalene, 7,10-14C-benzo[a]pyrene (51 mCi/mmol), and 2-14C-
phenobarbital (14 mCi/mmol) were purchased from Amersham/Searle, Des
Plaines, Illinois.
3-Trifluoromethyl-4-nitrophenol was obtained from Mr. John Howell, U.S.
Department of the Interior, Hammond Bay, Michigan. Radioactive 3-
trifluoromethyl-4-nitrophenol ( C ring uniformly-labeled, specific
activity, SA 3.7 mCi/mol) was obtained from the Mallinckrodt Chemical
Company, St. Louis, Missouri.
8-3H 5'-AMP (10 Ci/mmol) was purchased from New England Nuclear,
Boston, Massachusetts. C-(carboxyl label) phthalic anhydride (17.6
mCi/mmol) was purchased from ICN Pharmaceuticals Irvine, California.
Di-2-ethylhexyl (ring 14C) phthalate (10.52 mCi/mmol), I4C-(ring-UL)-
21
-------
pentachlorophenol (10.04 mCi/mmol) C-(Ring-UL)-l,2,4-trichlorobenzene, and
1,2,4,-trichlorobenzene were purchased from Pathfinder Laboratories,
St. Louis, Missouri.
Bentachlorophenol and naphthalene (certified grade) were obtained from
FisherScientific Company, Itasca, Illinois, and 2-methylnaphthalene (gold
label grade) from Aldrich Chemical Company, Milwaukee, Wisconsin.
Saffrole and tropitol were a gift from the McGormley-King Co.
(Minneapolis, Minnesota); piperonyl alcohol, piperonal, and 1,3-benzodioxole
were purchased from Aldrich Chemical Co. (Milwaukee, Wisconsin) and
piperonyl butoxide was purchased from Pfaltz and Bauer (Flushing, New York).
MEHP was a gift from Dr. David E. Stalling, U.S. Department of the
Interior, Columbia, Missouri.
Amberlite XAD-2 resin was obtained from Rohm and Haas Co.,
Philadelphia, Pennsylvania.
Tetrachloroquinone was donated by Dr. P. Gehring, Dow Chemical Co.,
Midland, Michigan, and tetrachlorohydroquinone was purchased from Eastman
Chemical Co., Rochester, New York.
Glass-distilled solvents were purchased from Burdick and Jackson,
Muskegon, Michigan. Precoated silica gel plates (Sil-G 25 and Sil-G 25 UV
254) were purchased from Brinkmann Instrument Company, Westbury, New York.
NCS tissue solubilizer and ACS scintillation mixture were purchased from
Amersham, Arlington Heights, Illnois.
C-labeled naphthalene and 2-methylnaphthalene were purified to
greater than 99% purity by TLC on me thanol -washed silica gel plates using
CCl^ or CHC13 as solvents. The ^C-trichlorobenzene was > 99% pure as
received.
Pentachloroanisole and C-pentachloroanisole were made by methylation,
respectively of pentachlorophenol and ^C-(Ring-UL)-pentachlorophenol with
diazomethane. The pentachloroanisole was purified by recrystallization from
hexane, and the ^C -pentachloroanisole purified by TLC on silica gel plates
with methylene chloride as solvent.
Di-2-ethylhexyl [carboxyl-* C] phthalate was prepared by refluxing
labeled phthalic anhydride with excess 2-ethyl-l-hexanol in benzene with a
trace of ^SO^ for 4 h. The solvent was removed by distillation, and
the l^C-DEHP was purified by TLC on silica gel in benzene :ethyl acetate
(19:1, v:v).
22
-------
SECTION 4
RESULTS
HEPATIC XENOBIOTIC METABOLIZING ACTIVITY IN RAINBOW TROUT
Fractionation and Subcellular Localization of Marker Enzymes in Rainbow
Trout Liver
Several reports have appeared on the metabolism of xenobiotics by fish
liver microsome*s. These studies, however, have utilized protocols developed
for studying liver microsomal metabolism in mammalian species. It is not
known, therefore, whether these supposed "microsomal fractions" are indeed
representative of the xenobiotic metabolizing enzymes of fish livers.
Studies were therefore initiated to characterize the subcellular fractions
obtained from a typical fractionation of rainbow trout liver homogenates
(Statham et al. 1977).
Rainbow trout weighing 80-100 g were obtained and used for the
preparation of liver subcellular fractions as described in Section 3.
Although the scheme described in Figure 1 was ultimately adapted to
fractionate rainbow trout liver into subcellular fractions, preliminary
experiments were carried out in an attempt to optimize conditions for a more
definitive separation of "marker" enzymes characteristic of various
subcellular components. Initially, a low speed spin at 120 x g for 5 min
was included in the scheme described in Figure 1 to remove intact cells, but
this resulted in variable distribution profiles, particularly for the plasma
membrane markers, alkaline phosphatase and 5'-nucleotidase. Thus, in all
the distribution profiles reported in this fractionation study, trout liver
homogenates were centrifuged at 600 x g for 20 min to obtain the nuclear and
cellular debris pellet (Figure 1).
Since the lysosomes are particularly important in protein and lipid
degradation in mammalian liver, an attempt was made to optimize
centrifugation conditions for a more definitive separation of the hydrolytic
enzymes in trout liver homogenates. Lowering of the force of the
mitochondrial spin or increasing that of the microsomal spin resulted in a
lowered yield (percent recovery) of the mitochondrial or microsomal marker
enzymes in their respective pellets without a significant increase of the
relative specific activity (R.S.A.) of acid phosphatase in the light
mitochondrial or lysosomal fraction.
Thus, in the standard fractionation scheme (Figure 1), the post
mitochondrial fraction was centrifuged at 13,300 x g for 10 min to obtain
23
-------
the light mitochondrial or lysosomal pellet. The subcellular distribution of
four hydrolytic or lysosomal marker enzymes in trout liver—fractionated
according to the scheme described in Figure 1—were studied and the results
reported in Table 1. These results show clearly that heterogeneity exists
in percent recovery and relative specific activity of the four hydrolytic
enzymes in the various subcellular fractions. 3-Glucuronidase and N-acetyl-
$-glucosaminidase yielded similar distribution profiles with most of the
particulate activity in the nuclear and mitochondrial pellets but with
little enrichment in the lysosomal fraction relative to the mitochondrial
fraction. Almost 50% of Cathepsin D activity was recovered in the nuclear
fraction. The R.S.A. of the enzyme was high and of similar value in both
the nuclear and lysosomal pellets, indicating enrichment in each fraction.
Acid phosphatase, a hydrolytic enzyme used most often to monitor lysosomal
enrichment in mammalian liver fractionation schemes, yielded R.S.A. values
that were similar for lysosomal and inicrosomal pellet, indicating a similar
enrichment of this enzyme in both fractions. Since the major goal of this
study was to explore the distribution profile of certain drug-metabolizing
enzymes in trout liver, which are known to reside in the microsomal fraction
of mammalian liver, further efforts in delineating a lysosomal fraction in
trout liver were not made.
TABLE 1. DISTRIBUTION OF HYDROLYTIC ENZYMES OF RAINBOW TROUT LIVER3
Mito-
Nuclear chondrial Lysosomal Microsomal Supernatant
pellet pellet pellet pellet 165,000x0^
Acid phosphatase (6)
Recovery, % 14.2 16.2 13.9 45.7 9.3
R.S.A. 0.8 1.3 1.8 2.1 0.2
$-Glucuronidase (4)
Recovery, % 25.2 27.2
R.S.A. 1.7 2.3
9.7
1.3
15.9
0.8
21.9
0.5
N-Acetyl-g - Glucosaminidase (5)
Recovery, % 26.7 25.6
R.S.A. 2.1 1.8
10.6
1.2
11.5
0.6
25.5
0.6
Cathepsin D (1)
Recovery, %
R.S.A.
47.2
2.0
14.4
1.1
12.8
1.9
18.3
0.9
7.2
0.2
1Figures in parentheses indicate the number of experiments in which the
enzymatic analyses were performed, and the values shown are averages of the
indicated number of experiments. Relative specific activity (R.S.A.) =
percent of total enzymatic activity:percent of total protein.
A full enzymatic characterization of subcellular fractions obtained by
the scheme outlined in Figure 1 was undertaken, and the results are
24
-------
summarized in Table 2. Approximately 70% of the succinic dehydrogenase was
located in the pellet obtained by centrifuging the post-nuclear supernatant
for 8,000 x g for 10 min. The R.S.A. of succinic dehydrogenase of this
fraction was 6.5, and the R.S.A. of the lysosomal fraction was 4.0,
indicating some contamination of the lysosomal fraction by mitochondria.
TABLE 2. ENZYMATIC AND PROTEIN ANALYSES OF SUBCELLULAR
FRACTIONS OF RAINBOW TROUT LIVER3
Mito-
Nuclear chondrial Lysosomal Microsomal Supernatant
pellet pellet pellet pellet 165,000x0^
Alkaline phosphatase (3)
Recovery, % 36.2
R.S.A. 2.0
5'-Nucleotidase (3)
Recovery, % 47.8
R.S.A. 2.4
Succinic dyhydrogenase (6)
Recovery, % 16.2
R.S.A. 0.6
Acid phosphatase (6)
Recovery, % 14.2
R.S.A. 0.8
Glucose-6-Phosphatase (6)
Recovery, % 12.1
R.S.A. 0.6
Lactate dehydrogenase (3)
Recovery, % 7.9
R.S.A. 0.4
4.0
0.3
6.0
0.6
67.6
6.5
16.2
1.3
12.0
1.1
2.0
0.2
3.0
0.4
4.1
0.6
16.2
4.0
13.9
1.8
10.5
1.6
1.8
0.3
36.5
1.7
31.4
1.4
0
—
45.7
2.1
64.5
2.9
2.6
0.1
20.2
0.4
10.5
0.3
0
—
9.3
0.2
0.9
—
85.6
2.2
Protein (8)
Recovery, %
20.3
11.8
7.5
22.8
37.5
Figures in parentheses indicate the number of experiments in which the
enzymatic and protein analyses were performed, and the values shown are
averages of the indicated number of experiments. Relative specific
activity (R.S.A.) = percent of total enzymatic activity:percent of total
protein.
Alkaline phosphatase and 5'-nucleotidase, markers of plasma membranes
in mammalian liver (Salyam and Trams 1972), yielded almost identical
distribution profiles. The percent recovery and R.S.A. values for these
enzymes suggest that plasma membranes of trout liver, under the
25
-------
homogenization conditions described, were localized mainly in the nuclear
and microsomal pellets (Table 2).
Of particular importance was the distribution of microsomal marker
enzymes in subcellular fractions of trout liver prepared according to the
scheme outlined in Figure 1. Glucose-6-phosphatase, a gluconeogenic enzyme
found in the microsomal fraction of mammalian liver (Solyam and Trams 1972),
was found to the extent of approximately 65% in the microsomal fraction of
trout liver (Table 2, Table 3). This enzyme was definitely enriched in the
microsomal fraction as indicated by the high R.S.A. of the fraction.
TABLE 3. DISTRIBUTION OF MICROSOMAL ENZYMES OF RAINBOW TROUT LIVERa
Mito-
Nuclear chondrial Lysosomal Microsomal Supernatant
pellet pellet pellet
pellet
165,000x9'av
Glucose -6-Hiosphatase (6)
Recovery,
R.S.A.
% 12
0
.1
.6
Rotenone-insensitive cytochrome
Recovery,
R.S.A.
NADPH (2)
Recovery,
J\» D • A.*
Glucuronyl
Recovery,
K.» t> • A«
Benzopyrene
Recovery,
£\« O • A«
% 14
0
% 13
0
transferase
% 17
0
hydroxylase
% 15
0
.1
.7
.4
.7
(2)
.8
.8
(2)
.9
.8
12.
1.
C
16.
1.
12.
1.
9.
0.
5.
0.
0
1
reductase
0
4
6
2
1
9
2
5
10.
1.
5
6
64.
2.
5
9
0.
-
9
NADH (3)
12.
2.
15.
3.
8.
1.
5.
0.
4
1
0
0
6
4
2
7
57.
2.
55.
2.
63.
2.
63.
2.
2
5
2
2
5
5
3
5
0.
0.
3.
0.
0.
1.
10.
0.
2
1
8
1
1
0
5
3
aFigures in parentheses indicate the number of experiments in which the
enzymatic analyses were performed, and the values shown are averages of the
indicated number of experiments. Relative specific activity (R.S.A.) =
percent of total enzymatic activity:percent of total protein.
Microsomal contamination of the high-speed supernatant was low, but
approximately 10-20% of the 5f-nucleotidase and alkaline phosphatase plasma
membrane markers were found in the 165,000 x g supernatant (Table 2). Assay
for lactate dehydrogenase indicated that approximately 85% of this enzyme
was recovered in the high-speed supernatant, indicating minimal
contamination of the membranous fractions by cytosolic material.
Since Table 2 is a composite of data accumulated over several
experiments, the results from a single subcellular distribution in which all
the enzymes and protein content were measured is presented in Figure 2. A
comparison of the relative specific activities of the marker enzymes from
26
-------
4
3
2
1
0
>- 4
> 3
i= 2
O
< 1
O 0
u.
O
Alkaline Phosphatase
N
r Acid Phosphatase
N
M
0 20 40 60 80 100
5' Nucleotidase
0 20 40 60 80 100
4 r Rotenone Insensitive
, NADPH CytochromeC
3 " Reductase
2
1
0
u.
i= 4
3
2
1
0
0 20 40 60 80 100
rSuccinic Dehydrogenase
0 20 40 60 80 100
4 r Rotenone Insensitive
NADH Cytochrome C
3 " Reductase
2
1
0 20 40 60 80 100
0 20 40 60 80 100
r -fl-Glucuronidase
Glucose -6-Phosphatase
0 20 40 60 80 100
0 20 40 60 80 100
Lactic Dehydrogenase
3
2
1
0
0 20 40 60 80 100
% TOTAL PROTEIN
Figure 2. Distribution of marker enzymes from trout liver.
27
-------
this individual fractionation with those presented in Table 2 indicates
excellent agreement.
After the fractionation scheme described in Figure 1 was characterized
with marker enzymes, the subcellular distribution of glucuronyl transferase
and benzopyrene hydroxylase in trout liver was explored. The results of
several distribution experiments focusing on microsomal enzymes are
presented in Table 3. Glucose-6-phosphatase, glucuronyl transferase, and
benzopyrene hydroxylase had the highest R.S.A. and percent recovery in the
microsomal fraction. Similar distribution profiles and R.S.A. were obtained
with rotenone-insensitive cytochrome C reductase, another microsomal marker
enzyme. However, with NADPH as co-factor, the high R.S.A. of the rotenone-
insensitive cytochrome C reductase in the lysosomal fraction indicated
significant contamination of this fraction by these enzymes. This was not
surprising in light of the report by Sottocasa et al. (1967) that this
enzyme also is located in the outer mitochondrial membrane.
In Figure 3 the results of one such distribution experiment are
illustrated. The relative specific activity profiles of glucuronyl
transferase and benzopyrene hydroxylase are almost superimposable with the
profile of glucose-6-phosphatase, a microsomal marker in mammalian liver.
Recovery of glucuronyl transferase and benzopyrene hydroxylase in the
microsomal fraction was approximately 70%, which was similar to that found
for glucose-6-phosphatase.
Although the results indicate that a sharp separation of all organelles
was not achieved in all cases, the procedure does allow for adequate
resolution of the major subcellular organelles. The non-homogeneity of the
lysosomal enzymes seen in this study has been observed in other systems,
and, since the major thrust here concerned microsomes, attempts were not
made to resolve lysosomes.
The R.S.A. and percent recovery of benzopyrene hydroxylase and
glucuronyl transferase were almost identical with these criteria for the
microsomal marker, glucose-6-phosphatase. The R.S.A. for rotenone
insensitive NADH and NADPH cytochrome C reductase indicates enrichment of
this activity in the lysosomal as well as in the microsomal pellet, but
these two fractions together contain 70-80% of all microsome markers.
Separation of microsomes from plasma membrane has been difficult to achieve
in other species, and the relatively high percentage (30-35%) of the plasma
membrane markers, alkaline phosphatase and 5'-nucleotidase, in the
microsomal fraction indicates that this same prpoblem also exists with trout
liver. However, the high R.S.A. of the plasma membrane markers in the
nuclear pellet indicates the greatest enrichment on a biochemical basis in
this latter fraction.
In several studies concerning the metabolism by liver subcellular
fractions of diazinon (Hogan and Knowles 1972), aniline (Pohl et al. 1974),
aldrin (Stanton and Khan 1973), and di-2-ethylhexylphthalate (Carter et al.
1974), a considerable amount of activity against these substrates has been
found to reside in the "mitochondrial" or 10,000 g pellet. The data
presented in this study indicate that, even under carefully controlled
28
-------
o
8
Succinic Dehydrogenase
> °
H
> 3
S 2
1
0
r Glucose-6-Phosphatase
CL 4
en
3
LJ
> 2
!< i
S °
3
2
1
0
Glucuronyl Transferase
• Benzopyrene Hydroxylase
0 20 40 60 80 100
% TOTAL PROTEIN
Figure 3. Distribution of mitochondrial and microsomal marker enzymes from
trout liver.
29
-------
fractionation procedures, some microsomes sediment with the mitochondrial
fraction and that biotransformation of these compounds by the mitochondrial
fraction is most likely associated with contaminating microsomes rather than
with mitochondria per se* This association is also shown in studies of di-
2-ethylhexylphthalate which utilized homogenization in 0.154 M KG1 rather
than the 0.25 M sucrose used in this study.
Since microsomes were the only liver subcellular fraction required in
the subsequent parts of Section 4 dealing with "hepatic xenobiotic
metabolizing activity in rainbow trout", the simplified fractionation
procedure described in Section 3 (Methodology Preparation of Subcellular
Fractions of liver) was utilized henceforth.
Effect of Polycyclic Aromatic Hydrocarbons on Hepatic Microsomal Enzymes in
Rainbow Trout
Now that the efficacy of the fractionation scheme for obtaining
microsomes from trout liver homogenates has been demonstrated, such
microsomes can be used in studies to characterize the xenobiotic
metabolizing activity of rainbow trout liver. Assays for a variety of
microsomal enzyme activities will be validated for use with trout liver
microsomes. The presence of these liver microsomal enzyme activities can be
evaluated in normal (control) trout and in trout exposed to agents known to
affect such enzyme activities in mammals.
The radioactive assays developed for arylhydrocarbon (benzo[a]pyrene)
hydroxylase (AHH) and UDPGA-gluruconyl transferase were linear with time and
protein concentration under the prescribed condition. The AHH activity in
hepatic microsomes of rainbow trout showed characteristics typical of
cytochrome P-450 mediated monooxygenation, i.e., the activity was dependent
on a source of NADPH and was inhibited by PBO, a classical inhibitor of
microsomal monooxygenations. The UDPGA-glucuronyl transferase activity was
negligible in the absence of UDPGA but was linear for up to 20 min in the
presence of UDPGA.
Three polycyclic aromatic hydrocarbons (g-naphthoflavone, 3-
methylcholanthrene, and 2,3-benzanthracene) were investigated for their
abilities to induce various microsomal enzyme activities in rainbow trout
(Statham et al. 1978). Figure 4 demonstrates that all three polycyclic
aromatic hydrocarbons caused dramatic increases in AHH activity without
affecting glucose-6-phosphatase or UDPGA-glucuronyl transferase.
Time-course studies for the induction of AHH by the polycyclic aromatic
compounds showed a typical time-dependent increase in monooxygenase activity
(Figure 5). The AHH activity reached a maximum at about 48 h after
injection of 3-methylcholanthrene and 2,3-benzanthracene, but with g-
naphthoflavone as the inducer the AHH activity was still increasing 96 h
after injection. These differences may be explained by differences in rates
of absorption of the various compounds from the intraperitoneal corn oil
depot into the vascular system of the fish. Similar observations by Boobis
et al. (1977) have been explained in the same manner. However, in mice, it
was found that AHH activity returned to basal levels faster after g-
30
-------
5
ft
s
o
E
14-
12-
10-
8-
6-
4-
2-
0
GLUCOSE-6- PHOSPHATASE
ffl
00'
o
u_ c-g
O
UJ
Q_
CO
1.0-,
0.8-
0.6-
0.4-
0.2-
X
C
F
0
0
0
0
0
0.
0
0
8 -,
.7-
6-
.5-
4-
3-
2-
1 -
0
GLUCURONYL TRANSFERASE
J?L
BENZOPYRENE HYDROXYLASE
T
T
C ^ T
'BENZANTHRACENE
3- METHYLCHOLANTHRENE ^-NAPHTHOFLAVONE
Figure 4. The effect of various inducing agents on selected microsomal
enzyme activities. Animals were pretreated as described in
Section 3 (Methods). Hepatic microsomes were prepared from
individual fish 48 h after dosing. Each bar is the mean ± S.E.
(n = 3-5).
* Indicates induced activity (T) significantly different from control
activity (C) (p < 0.05).
31
-------
c
o
+•>
o
0^
IE?
i_
Q)
+J
S-
TO
V)
u.
3
O
X
euaiAd [e] ozueq
O 0)
ft &
4J
co
3 (50
O f3
M CO CU
cd £3 fi
> Q)
& O
>1 Cy> CO
,O Ct) M
0) rC
co co a
cd 4-> cd
H p3 N
>, cy o
X a -> co
ctf CO i-H
•—• O
O O t-l
N J? 4J
C 4J C
Q) O
,£14-10
<^ O
iH Q)
•H C
O O
a
o
cd M H
O 0) O M-l
o rc: a o
t-l 4-> ,C
CO
i
Cd T-l
o
M-l P.
•§.
f
ca.
a o
o cd
• ft
J3 D
CD T
•H JL
M-l O
a
1
•H O
4-1 01
o cd C
M-I O co cd
O t-l iH
cu vj cu o
CO T3 > jS
t-l >~i -H O
3 .Ti r-l H
o >,
CJ O T3 ,C
I T-l 0) 4J
CU r-l rH CU
0 O O 0
•H >, O I
H CJ O. rO
M
•H
32
-------
naphthoflavone pretreatment than after injections of 3-methylcholanthrene
(Boobis et al. 1977).
The induction of AHH by 3-methylcholanthrene, 3-naphthoflavone, and
2,3-benzanthracene was accompanied by an increase in levels of hepatic
microsomal cytochrome P-450 (Table 4). The P-450 hemoprotein concentrations
of the induced microsomes were approximately 50% higher than those of the
control microsomes.
TABLE 4. CYTOCHROME P-450 CONTENT OF
VARIOUSLY-INDUCED TROUT HEPATIC MICROSOMES
Pretreatment of fish
Corn oil (control
3 -Naphthoflavone
3-methylcholanthrene
2, 3-Benzanthracene
Dose,
mg/kg
1
100
20
10
Cytochrome P-450,
nmol/mg of protein
0.210; 0.223a
0.334; 0.354a
0.311; 0.337a
0.336; 0.277a
aValues obtained from pooled microsomes (6-8 fish/group) in two separate
experiments. Microsomes prepared 48 h after treatment of fish.
Polycyclic aromatic hydrocarbon inducing agents have been shown
previously to induce AHH activity in several marine species (Bend et al.
1973, James et al. 1977); in these studies, however, no concomitant increase
in cytochrome P-450 levels were observed. In the present studies—using
rainbow trout—increases in cytochrome P-450 concentration in response to
3-naphthoflavone, 3-methylcholanthrene, and 2,3-benzanthracene were found.
This increase in cytochrome P-450 levels may reflect differences in the
control mechanisms of P-450 systhesis of marine and freshwater species of
fish.
No distinctive alterations in the position of the Soret peak of the CO
complex of the NaSOoO^-reduced hemoprotein was noted after induction by the
polycyclic hydrocarbons. This finding contrasts with the situation in rats
and other mammals where a shift from 450 to 448 run was observed (Sladek and
Mannering 1966).
Further evidence for a true induction, rather than an activation of
existing enzymes, was found in in vitro studies. The inclusion of
3-naphthoflavone in the AHH assay, or the preincubation of control
microsomes with 3-naphthoflavone and NADPH prior to the AHH assay, did not
increase the hydroxylation of benzo[a]pyrene. The data in Table 5
demonstrate that 3"naphthoflavone was actually inhibitory to the AHH assay.
The observation that 3-naphthoflavone, added in vitro, inhibited the
AHH activity of control microsomes suggests that the endogenous cytochrome
P-450 of trout hepatic microsomes may be of the P,-450 type. This
33
-------
suggestion seems likely because in mammalian systems g-naphthoflavone
actually stimulates P-450 mediated AHH while inhibiting i^-450 mediated AHH
activity (Wiebel et al. 1971). Furthermore, no blue shift in the absorbance
TABLE 5. IN VITRO EFFECT OF g-NAPHTHOFLAVONE
ON ARYLHYDROCARBON (BENZOfA]PYRENE) HYDROXYLASE
ACTIVITY OF HEPATIC MICROSOMES IN CONTROL TROUT
Addition to assay
None
Dimethylformamide
g -Naphthof lavone13
5 y liter
10 \iM
100 \iM
500 yM
AHH activity3, %
100
98.3
65.3
46.3
22.6
anmol of polar metabolites of benzo[a]pyrene produced/min/mg microsomal
protein.
g-Naphthof lavone was added dissolved in 5 yl of ^W-dimethylformamide.
maximum of the CO complex of the ferrocytochrome was seen after induction,
which suggest that the induced and endogenous cytochrome(s) are similar.
These differences between the induction of hepatic microsomal
cytochrome(s) P-450 in fish and in mammals indicated the need for a more
detailed study of induction and cytochrome(s) P-450 in rainbow trout.
Induction and Characterization of Cytochrome(s) P-450 and Ifcmooxygenation in
Rainbow Trout
The four model monooxygenase assays were validated in rainbow trout
hepatic microsomes (Elcombe and Lech 1979). Temperature optima of 25-26°C
were found for ethylmorphine-W-demethylation and benzo[a]pyrene
hydroxylation, while for ethoxyresorufin- and ethoxycoumarin-0-deethylations
the optima were 29-31°C. All reaction velocities were calculated over the
linear portion of the reaction. The dependence of the reaction velocities
on the concentration of microsomal protein was also examined. These results
are depicted in Figure 6. These parameters were constant for all rainbow
trout hepatic microsomal preparations examined.
Pretreatment of rainbow trout with the polyhalogenated biphenyls
(Firemaster BP6, Aroclor 1242, or Aroclor 1248) had no effect on the
liver/body ratios or on the yields of microsomal protein per unit wet weight
of liver (Table 6) (Elcombe and Lech 1973). However, PBBs and PCBs were
able to induce the activity of certain monooxygenase reactions.
Dose-response studies of the induction process indicated maximal
induction of PBBs and PCBs at about 250 mg/kg (Figure 7 and Figure 8).
34
-------
-i CM
c
'cu
-t-l
e
Q.
"TO
I
s
O
'£
CJO
O
uiiu/ui.jJuosaj IOLUU
CM
uiuu/oHOH
en
I
o
en
o
o
•H
B
o
•H
4-1
td
o
f-i
4-1
§
rd
C
•H
rt
O
•H
4-1
CO
a
cu
60
o
o
c
CO
o
•H
S-l
n)
o
fi
a;
T3
g
ex
Q)
a
•rl
CU
4J
O
i-t
PM
60
•rl
35
-------
CO
cu
s
o
co
o
M CU
CU CO
4J Cfl
H-l i-l
BUI-UIU
co co o
CU >> JJ
3 cfl -O
i-H T3 >•>
Cfl J3
!» »*
0
O
CO
1-1 1-1 M
CU T-l cfl
CO 0 O
cu fi o
h CU M
cu eu >-.
4-1 Q) >>
a M i-i
•H CU Cfl
CN
co
f, CU
O 3
cfl r-l
W cfl 0
>^
4J
Cfl
•a- ,c rH
rg co >-,
H >H ^S
M-l 4-1
M CU
O X CU
H -H T3
O CO I
O ^>
•H
»-< co m
o M 3
m cu M
> o
•H co
Cu .—I 0)
•H M
X T3 >-,
W CO X
C H Q
O O J3
rH CU
CU J3
5-1 t!
pnpoJd |ouiu
cu B
co o
C M
O M-l
a.
en T3
cu cu
1-1
0) CU
co co
O ,0
a o
w
> 4-J
M <-»
cu
M
60
•H
36
-------
co
o o
CO -H
O W
H O
O CO
1!
t-i CD
O)
_*
\
O>
E
O
Q
co cd
cu
3 co
rH >, •
cd to a)
> TJ co
a)
CO -vf r-t
C T3 ^
CU Q) O
CO (3 H
-,
Cd 0) ^3
1
PH
«
14 CO
CU
.[ j n i
co •"
S -H O
CU CO -H
lj Jj
•H M-l Cd
PH O H
>.
CU
>
CX.
•H
co
fi .
O
cu
co
O
&
CO
o
co
cu
>>
o
4-1
cu
I -H
uiejojd OJDIUI Biu-uiui
* • • •
4?npojd |Oiuu
I -H A
CU 0} T
CO W
O 3 I
00
0)
1-1
3
00
•H
37
-------
TABLE 6. LIVER: BODY WEIGHT RATIOS AND YIELD OF MICROSOMAL PROTEIN3
mg microsomal
Treatment Dose Liverrbody ratio, % protein/g liver
Corn oil
Firemaster BP6
Aroclor 1254
Aroclor 1242
1 ml /kg
150 mg/kg
150 mg/kg
150 mg/kg
0.96 + 0.05
1.07 + 0.04
0.92 + 0.03
0.96 + 0.06
24.5 + 0.8
24.0 + 0.5
25.0 + 1.6
23.6 + 2.7
lValues are mean + SE (n=6).
Aroclor 1242 maximally induced arylhydrocarbon (benzo[a]pyrene) hydroxylase
(AHH) activity by approximately 10-fold, while PBBs resulted in only a
4-fold increase. Ethoxyresorufin-O^deethylase was maximally stimulated by
approximately 30- and 20-fold by PBBs and Aroclor 1242 respectively.
Studies of the time-course of induction for Arodors 1242 and 1254 at a
dose of 150 mg/kg indicated that maximal stimulation of monooxygenation was
attained after 4 and 7 days respectively (Figure 9 and Figure 10). A single
intraperitoneal injection of Aroclor 1242 elevated AHH, ethoxycoumarin-i9-
deethylase, and ethoxyresorufin^O-deethylase by about 10-fold at 4 days
post-injection. These enzyme activities remained elevated for at least 15
days after treatment of the fish (Figure 9). Figure 10 shows a similar 10-
fold induction of monooxygenase activity by Aroclor 1254. Stimulation of
monooxygenation was still apparent 21 days after treatment.
Ethylmorphine-#-demethylation was unaffected by pretreatment of fish
with Aroclors 1254 or 1242. This finding is illustrated for Aroclor 1254 in
Figure 10.
After injection of rainbow trout with Firemaster BP6, no increases in
monooxygenase activities were seen until 3 days after treatment (Figure 11).
After this time, ethoxyresorufin- and ethoxycoumarin-tf^deethylations
remained elevated for at least 2 weeks, although AHH declined rapidly to
reach control levels about 7 days after treatment of the fish.
Ethoxycoumarin-0-deethylation did not reach a maximum until 7 days after
injection. This delay has also been observed in rats treated with
Firemaster BP6 (Dent, et al. 1978).
In common with the PGBs, PBBs also failed to increase ethylmorphine-#-
demethylation above control values. In general Aroclor 1254 and Aroclor
1242 appeared to be more potent (in terms of mg/kg) than Firemaster BP6;
however, the latter compound was much more effective at inducing
ethoxyresorufin-tf -deethylation.
The stimulatory effect of PCBs and PBBs on monooxygenations appears to
be a true induction since inclusion of these compounds in the i,n vitro
assays had no effect upon the observed enzymatic activities.
38
-------
o
Q
UlOJOJd OJ3JUI BUMIJUI
JouuJ
3
60
•H
39
-------
S
jouiu
a
-H
cu o i
G f. fes
O 4-1 I
4-1 CU CU
•H C
cn
cu
CU
ft
co
M
i
•H C >>
O ,C
CO TJ -H 4-1
> CU W CU
CO CU iH •
4-1
cn
0)
to
0)
4J
cu
cu
ft -X3 I CU
CU CO C5 CO
M I CO
CO C rH
4J tO 'H >,
£2 !3 14-1 *
3 O
O
ft
O
CO
O T3
CN CO >~,
cu ^
>v C
w o x o
rH O fi
• O 4J CO
<3- J_l CU CJ
LO <^ O
CN .. J-l
o cn -IT i—i
>H -H ^
O 14-1 VJ
O • CO
M X M
•< -H ,i M-l -H
3 -H O 4-J
13 i-l CO
C CU rH
•H cu cn >-,
r*] Q pj
M 4-1 T3 4-J
o cu
M-l 0 CB CU
O ID •
0) tH 4-1 I CU
cn MH cfl •
O C3 O ^4 rC!
I -rt CB 4J
cu co C S
e 4-i ^ 3
O O
0)
H O O O
cu
M
bO
•rl
40
-------
CO
1
co
o
M
O
•H
S
op
6
OJ3IUI 6ui>uui
14-1 T3
CU •"
CO rl C
cu cu o
3 4J -H
rH CO 4-1
cfl -H Cfl
> C rH
CO B r3
4J T3 4-1
C Cfl CU
CU CU •
CO CO T3 CU
CU Cfl I CO
ft ^ 7 rH
PQ 14H O
4-1 3 M
C t-l (-1 TJ
•H CU O f>^
O 4J CO ,£
A, CO CU
^2 B t^ O
O CU X rQ
cd j-i o i-i
W -H ^3 cfl
CU O
PQ CO
•H
l^ l[ I
CU
en -H
f> -H rH
C MH
o
•rl
0 •
• »t
O (3
o
-rl
O O
3 O
cu ,d
CO 4-1
o cu
TJ CU
•H CU
^3
M W
>
4-1 X
•H O
•rl fl 0) 4J
H O A, CU
o cu
I -H
CU ct)
cu
VJ
60
•rl
41
-------
Hemoprotein P-450 concentrations in rainbow trout hepatic microsomes
were determined 4 days after pretreatment with various inducing agents.
Small increases (10-20%) in hemoprotein P-450 content were observed after
treatment of fish with PCBs or PBBs, but these increases were not
statistically significant (Table 7). However, pretreatment of fish with $-
naphthoflavone resulted in a significant 40% increase in the level of P-450
hemoprotein.
TABLE 7. CYTOCHROME P-450 CONTENT AND 455/430 PEAK RATIOS OF EtNC SPECTRA
Treatment Dose P-450 EtNC spectra
nmol/mg protein 455/430 ratio
Corn oil
Phenobarbital
Fi remaster BP6
Aroclor 1254
Aroclor 1242
3 -naphthoflavone
1 ml/kg
65 mg/kg
150 mg/kg
150 mg/kg
150 mg/kg
100 mg/kg
0.118 + 0.003
0.111 + 0.006
0.132 + 0.014
0.143 + 0.020
0.137 + 0.001
0.165 + 0.005a
0.28 + 0.01
0.32 + 0.04
0.37 + 0.05
0.35 + 0.05
0.39 + 0.01
0.40 ± 0.10
aSignif icantly different from corn oil
determination used pooled microsomes
control group, p < 0.
from the livers of two
05, n = 3. Each
fish. Values
are mean 4- SE.
Significantly, the A for the CO complex of reduced cytochrome P-450
was at 449 nm in all preparations examined (Figure 12).
The interaction of ethylisocyanide with Na2S£0^ reduce cytochrome P-450
resulted in optical difference spectra with absorption maxima at 432-433 nm
and 453-454 nm in all microsomal preparations examined. A small increase in
the 455:430 peak ratio was observed after pretreatment with PBBS, PCBs, or
3-naphthoflavone (Table 7).
The inducing properties of the polyhalogenated biphenyls appear to be
expressed differently in fish than in rodents. The polyhalogenated biphenyls
failed to increase liverrbody weight ratios or yield of microsomal protein;
furthermore, increases in ethylmorphine-^-demethylase were not apparent.
These responses are typical of polyhalogenated biphenyl induction in rodents
(Ecobiochon and Comeau 1974, Bickers et al. 1975, Goldstein et al. 1975,
Dent et al. 1976a, 1976b, 1977a) and are characteristic of cytochrome P-450
induction caused by phenobarbitals. PCB and PBB treatment of rainbow trout
were found to stimulate AHH, ethoxycoumarin-0-deethylation, and
ethoxyresorufin-0-deethylation. AHH and ethoxycoumarin-0-deethylation are
induced by both phenobarbital and 3-methylcholanthrene in rodents; however,
ethoxyresorufin-0-deethylation is specifically inducible by the polyclic
aromatic hydrocarbon type of inducing agents (cytochrome P,-450) (Burke and
Mayer 1974).
42
-------
O12-
O-O8-
-OO4
O-O4-
425 45O 475
wavelength nm
5OO
Figure 12. Hemoprotein P-450 difference spectra. Mlcrosomes (24 mg/ml) were
divided between two cuvettes. A baseline of equal light
absorbance was obtained and CO was bubbled through each cuvette.
A few mg of NaaSaOit were added to the sample cuvette, and the
resultant spectrum between 420 and 510 nm was recorded.
3-naphthoflavone (BNF) (100 mg/kg); Aroclor 1254 (A1254) (150
mg/kg); Aroclor 1242 (A1242)(150 mg/kg); Firemaster BP6 (FBP6)
(150 mg/kg); corn oil (C) (1 ml/kg).
43
-------
Because of the ability of commercial polyhalogenated biphenyls to
elicit both phenobarbital-like and 3-methylcholanthrene, "mixed" inducer has
frequently been used. Other studies have demonstrated that cytochrome P, -
450 or cytochrome P-450 induction by PCBs is determined by the substitution
pattern of the halogen atoms (Goldstein et al. 1977, Poland and Glover
1977). More recently, Moore and Aust (1977) and Moore et al. (1977) have
suggested that a similar situation exists with the PBBs.
In contrast to the apparent "mixed" patterns of stimulation of
monooxygenation in rodents due to polyhalogenated biphenyls, rainbow trout
seem incapable of responding to induction of cytochrome P-450 related to
phenobarbitals.
The large increases (up to 25-30 fold) in monooxygenase activity
observed after PCS or PBB treatment of fish cannot be explained in terms of
an increase in concentrations of total hemoprotein P-450, since the levels
of this (these) enzyme(s) were increased by only about 10-20%. Hence, a
novel enzyme with different substrate specificity is possibly induced by the
PBBs and PCBs in rainbow trout. However, no changes in the wavelength of
the absorption maximum of the CO complex of ferrocytochrome P-450 were seen,
nor were the ratios of the 455:430 nm absorption maxima of the
ethylisocyanide complex of ferrocytochrome P-450 significantly altered. In
rodents the 455:430 peak ratio increases considerably after treatment with
PCBs or PBBs (Alvares et al. 1973, Goldstein et al. 1977, Dent et al. 1977).
Dose-response studies for induction of arylhydrocarbon (benzo[a]pyrene)
hydroxylation by (3-naphthoflavone indicated that maximal induction was
obtained using a dose of 100 mg/kg (Figure 13) (Elcombe and Lech 1979).
To obtain more detailed biochemical results on the properties of
induced hemoprotein P-450-mediated reactions, 3-naphthoflavone and Aroclor
1242 were used in doses of 100 and 150 mg/kg, respectively. The dose of
phenobarbital was 65 mg/kg since preliminary studies indicated that a dose
of 80 mg/kg was toxic to a majority of fish. Animals were sacrificed and
microsomes obtained 3 days after injection.
Table 8 demonstrates that none of the pretreatments had effects upon
either the liver:body weight ratio or the yield of microsomal protein
obtained from rainbow trout liver.
Hepatic microsomal ethylmorphine-#-demethylation was unaffected by
pretreatment of rainbow trout with g-naphthoflavone, Aroclor 1242, or
phenobarbital; however, arylhydrocarbon (benzo[a]pyrene) hydroxylation was
increased 10- and 40-fold by Aroclor 1242 and g-naphthoflavone,
respectively. Phenobarbital had no apparent effect upon arylhydrocarbon
hydroxylase activity (Table 9).
Michaelis-Menten kinetics for the deethylations of ethoxycoumarin and
ethoxyresorufin were examined in the variously-induced trout hepatic
microsomal preparations. Figure 14 indicates the biphasic nature of the
Lineweaver-Burk plots obtained for ethoxycoumarin-0-deethylation in
microsomes from g-naphthoflavone, Aroclor 1242 pretreated, and control
44
-------
d
o
•H
•u
ctf
O
M
TJ
01
d
0)
o
N
d
OJ
O
•H
•a
0)
d
o
JJ
JS
a
QQ.
o
M-l
CO
d
o
•H
4J
cfl
uid4Ojd OJDIUJ Bui'Ujui
sqoiaui audMd(D)ozuaq jotuu
3
O
o »
P, O
CO rQ
-------
c
'w.
1
1
1
T3
fsl
0
E
c
c
0)
o.
d
b
e
£
X
c
e
160
140
120
100
80
60
40
20
Control
Arochlor 1242
Ethoxycoumarin
0.15 0.20
Figure 14. Lineweaver-Burk plots for ethoxycoumarin-0-deethylation by
rainbow trout hepatic microsomes. 3-NF is 3-naphthoflavone.
A-A, Corn oil-treated fish; O-O, Aroclor 1242-treated fish;
• -•, 3-naphthoflavone-treated fish.
46
-------
TABLE 8. MICROSOMAL YIELDS AND LIVER:BODY RATIOS AFTER
PRETREATMENT OF RAINBOW TROUT WITH VARIOUS INDUCING AGENTS3
Pretreatment
Corn oil
Aroclor 1242
3 -Naphthof lavone
Phenobarbital
Dose
1 ml/kg
150 mg/kg
100 mg/kg
65 mg/kg
Microsomal yield,
mg protein/g liver
14.21 ± 0.38
15.00 ± 2.33
13.49 ± 0.37
14.95 ± 0.04
Liver/body ratio,
0.99 ± 0.03
0.99 ± 0.05
1.00 ± 0.01
0.96 ± 0.08
aValues were determined 72 h after injection of fish and are mean ± SE.
TABLE 9. INDUCTION OF MONOOXYGENATION IN RAINBOW TROUT HEPATIC
MICROSOMES FOLLOWING INTRAPERITONEAL PRETREATMENT
Ethylmorphine-^^demethylase
Pretreatment
Corn oil
1 ml/kg
Aroclor 1242
150 mg/kg
g -Naphthof lavone
100 mg/kg
Phenobarbital
65 mg/kg
nmol/min/mg
0.768 ± 0.054
0.753 ± 0.067
0.678 ± 0.137
0.622 ± 0.095
% control
100
98
88
81
Benzo [a ] pyrene
nmol/min/mg
0.022 ± 0.003
0.233 ± 0.012a
0.898 ± 0.119a
0.023 ± 0.004
hydroxylase
% control
100
1059
4081
104
aSignificantly different from control (corn oil) group. Values were
determined 72 h after injection of fish and are mean ± SE.
trout. In Figure 15, the ordinate has been expanded for clarity. The
double reciprocal plot obtained for the deethylation of ethoxycoumarin by
phenobarbital-microsomes was identical to that of the control microsomes.
Ethoxyresorufin-tf-deethylation depicted typical linear Lineweaver-Burk
plots in all cases (Figure 16).
Table 10 summarizes the kinetic parameters obtained for the
(9-deethylation reactions in variously-induced microsomes. The apparent Km
values for ethoxyresorufin-0-deethylations (about 150 uAf) were unchanged by
pretreatment of the rainbow trout. However, pretreatment with Aroclor 1242
and g-napthoflavone resulted in 13- and 44-fold increases in the apparent
V x values for the reaction. 3-Napthoflavone and Aroclor 1242 increased
the apparent Vmax values for ethoxycoumarin-O^deethylation by about 11- and
3-fold, respectively; these agents also decreased the apparent Km values
from 129 to about 50 uAf.
47
-------
0>
I
CO
o
bO
•H
48
-------
o
CO
o
_\ peuijoj. uij.ruos9j puuu
cfl
p.
4-1
3
o
o
•§
•H
rt
o
•H
4-1
cti
0)
0)
T3
Ci
14-1
3
M
o
CO
0)
S-l
JJ
0)
S-i •
O 0)
y-i a
co >
4J CB
O -H
iH
-------
TABLE 10. THE EFFECT OF INDUCERS ON THE KINETICS OF MONOOXYGENATION
IN RAINBOW TROUT HEPATIC MICROSOMES FOLLOWING INTRAPERITONEAL PRETREATMENT
Ethoxyresoruf in-(9-deethylase Ethoxycoumarin-<9-deethylase
Vmax % Km,
Pretreatment nmol/inin/mg control r\M
Vmax %
nmol/min/mg control
0.136±0.026a
Corn oil
1 ml/kg
Aroclor 1242
150 mg/kg 1.85l0.04b
3-Naphthoflavone
100 mg/kg 6.0610.18b
Phenobarbital
65 mg/kg 0.08810.017
100 144±6 0.10110.010 100 129±9
1367 15410 0.28610.047a 283 5111l
4455 12518 1.1910.283 1178 4114*
65 17010 0.06510.017 64 10515
Values are mean ± SE; all values obtained 72 h after injection of fish.
'Significantly different from corn oil control group, P < 0.05.
Inhibitor Studies
Additional information on the nature of the cytochrome(s) P-450 present
in trout liver microsomes can be gained through use of inhibitors in
vitro* Previous studies in rodents, have shown that is is possible by the
differential inhibition of microsomal monooxygenation to characterize
cytochrome(s) P-450 subpopulations. 3-Naphthoflavone a potent inhibitor of
cytochrome P -450 in rodents, almost completely abolished arylhydrocarbon
(benzo[a]pyrene) hydroxylation in microsomes obtained from control,
g-naphthoflavone-, Aroclor 1242-, and phenobarbital-pretreated trout (Table
11). Metyrapone, an inhibitor of cytochrome P-450 in rodents, had little
effect on the arylhydrocarbon hydroxylase activity in control,
3-naphthoflavone, Aroclor 1242, or phenobarbital microsomes.
The hemoprotein P-450 content of hepatic microsomes from control fish
was 0.234 nmol/mg protein and the absorbance maximum of the
carboxyferrocytochrome complex was at 449 nm. 3-Naphthoflavone and Aroclor
1242 increased the specific content of P-450 to 0.341 and 0.261 nmol/mg
protein, respectively (Table 12); however, these agents did not affect the
position of the Soret absorbance maximum (Figure 12). Phenobarbital had no
effect on these parameters.
Ethylisocyanide (EtNC) reacts with ferrocytochrome(s) P-450 to produce
a complex that exhibits absorbance maxima at about 430 and 455 nm. The
ratio of these peaks has been used to distinguish between different forms of
hemoprotein(s) P-450. A slight increase in the 455:430 ratios was observed
after pretreatment of trout with Aroclor 1242 and 3-naphthoflavone
50
-------
TABLE 11. THE IN VITRO EFFECTS OF g-NAPHTHOFLAVONE AND METYRAPONE
ON ARYLHYDROCARBON (BENZO[a]PYRENE) HYDROXYLASE IN HEPATIC
MICROSOMES OF VARIOUSLY-PRETREATED RAINBOW TROUT
Inhibitor
None
Metyrapone
a-Naphtho-
flavone
Concentrate
\iM
Pretreatment of fish
°n> Corn Aroclor g-Naphtho-
oil Phenobarbital 1242 flavone
100b 100
(0.024)c (0.026)
10
100
500
10
100
500
96
100
85
19
15
12
92
77
84
23
14
9
100
(0.757) (1
116
85
75
22
9
9
100
.003)
96
88
84
9
2
1
a Inhibitors were added dissolved in 5 pliter Nf tf-dimethylformamide.
solvent had less than a 10% effect on the enzymic activity.
Values are percentage of remaining activity.
GValues in parenthesis are activity expressed as nmol/min/mg protein,
This
»
TABLE 12. CYTOCHROME P-450 CONTENT AND 455:430 PEAK
RATIOS OF EtNC SPECTRA
Pretreatraent
Corn oil
Phenobarbital
Aroclor 1242
g-Naphthof lavone
Dose
1 ml/kg
65 mg/kg
150 mg/kg
100 mg/kg
EtNC Spectra,
455:430 ratio
0.28 ± 0.01
0.32 ± 0.04
0.39 ± 0.01
0.44 ± 0.10
P-450,
nmol/iag protein3
0.234 ± 0.006
0.222 ± 0.010
0.261 ± 0.001
0.341 ± 0.010b
^Assuming extinction coefficient of 100 tnW cm .
Significantly different from corn oil control group, p < 0.05. Values are
mean ± SE, n = 3; each determination used pooled microsomes from the
livers of two fish.
(Table 12); however, the increase was not statistically significant at the
level of significance chosen in this study.
This study, in contrast with previous reports, demonstrated that type I
and type II compounds would elicit binding spectra with rainbow trout
hepatic microsomal hemoprotein(s) P-450. Figure 17 and Figure 18 illustrate
51
-------
!
I
CD
o
5
R
CO
o
%C
13
•rl M
cd e
42
•U >,
•rl rH
> CU
cu cd
•H -rl
X X
O O
4-1 1-1
O
O
in cu o
rH O
C p. m
cu a
cu cd 0
& to cu
0) CU &
43 42 4J
4-1 rl
T) Cd
cd
4J CU
43 Id
o nd
cd TJ
cu cu
o cu Td
C n M
cd cu o
43 &
o
to
(1)
o to • ^
p. oo 3
•H rH -H rH M
P. O rH Cd 4J
M 4-1 U
-d 4J rH -H 0)
C C Cd 43 P.
cd O 3 H co
o cr cd
cu 42 cu
O 43
5 >*H X H
M O CU
cd B
cu
G
O -H rH
•H O
0)
MH
O
cd
V-i
4J
O
cu
&, CO
to O
oo o
to
fi
cu
f^ O
to
3 cd
to
Td
rH (3
cd cd
CO
m
oo
C
O
0)
4-1
cd
0)
co u
• o
to £
CU 4-1
S P
o c -H
CO CU P-i
O Q)
13
_
iH
S
o Td o
•H cu m
4J Td PO
cd «H
^.^ "2
43 TJ cd
S
•H
M-l
cd S
cu o
4-i in
4J ro
cu
^ 'a
3 a
o cd
cu
M
3
52
-------
fl
• oo
CO
a) m
B O
O
co d
o o
H -H
O 4->
•H cd
e M
4-1
o d
•H Q)
4-1 U
cs d
P. O
cu o
m
3 C
O -H
o
& 4-1
•H T)
0) 0)
•S !
0)
rH (1)
O rH
N O
0) N
T3 tfl
I
O 4J
cu
O
O
Q) A
G.LO
CO
cu
00 M
d 3
•rl 60
T3 -H
d PH
•H
R
n
d co
tO cd
M 4J
•H
> O
H O
oo
00
•H
53
-------
typical binding spectra for PBO (type I), hexobarbital (type I), and
imidazole (type II).
Furthermore, the formation of methylenedioxyphenyl metabolic
intermediate-ferrohemoprotein P-450 complexes, which is seen with rodent
microsomes, also was observed using rainbow trout hepatic microsomes (Figure
19).
Uptake and Elimination of Phenobarbital from Liver
The lack of effect of phenobarbital upon monooxygenase activity could
be due to poor adsorption of the compound from the intraperitoneal cavity.
However, studies utilizing C-phenobarbital suggested that this was not the
case (Figure 20). Uptake of the compound was rapid, and elimination of
radioactivity followed a time-course similar to that observed in rodents.
Effect of Pretreatment Agents upon In Vitro Arylhydrocarbon
Hydroxylation—
The observed increases in monooxygenase activity after pretreatment of
trout with 3-naphthoflavone or Aroclor 1242 possibly was caused by an
activation rather than an induction phenomenon. Similarly, the lack of
response to phenobarbital may have been due to inhibition of monooxygenation
by the barbiturate, as suggested by previous studies (Bend et al. 1973).
However, studies of arylhydrocarbon (benzo[a]pyrene) hydroxylation in the
presence of various concentrations of these agents (10-500 yAf) did not
support these suggestions. Table 13 illustrates that Aroclor 1242 had
little effect upon monooxygenation in vitro but that 3-naphthoflavone
dramatically inhibited the reaction similarly to the effect of g-
naphthoflavone (Table 11). Phenobarbital had little effect upon the
arylhydrocarbon hydroxylase activity in vitro,
Discussion—
Monooxygenase activities of untreated rainbow trout hepatic microsomes
generally were lower than in many rodent species. For comparison, Table 14
shows representative values from our laboratory for mammalian
monooxygenation reactions. Ethoxycoumarin-tf-deethylation (Vmax=0.101
nnol/min/mg) and ethoxyresorufin-0-deethylation (Vmax=0.136 nmol/min/mg) in
trout were lower than the corresponding mammalian values. However, these
values do not reflect a less active hemoprotein P-450, since a 3-4 fold
difference in hemoprotein level exists and since, when activity is expressed
per unit of hemoprotein, little if any difference exists. Considering the
lower concentrations of hemoprotein, trout have significantly higher
ethoxyresorufin-<9-deethylase activity than mammalian species. Similarly,
benzo[ajpyrene hydroxylase activity is several-fold higher in trout than in
rats (Ahokas et al. 1976, 1977).
54
-------
_R
-8
0,5
CO
o
Q) O
o
I
•s
•H
rt
co S3
rt PM
!» Q
p. -u
01
M
3
53
-------
I
cd
^
4-1
•rl
I
o
I
m
00
O
,.,
rl
o) co ca
4J rH
o cfl iw
cu > o
I? cu M
•H 4-J 01
a >
CU iH -H
l-i rH
CU 4->
£ rt 01
43
x-v t3 4-1
60 0)
'—f G
O H -H
O -H
O t3
00 C
4-1 /-\
3M
O ^
^Q O CU
•H I
• 60
1-t 0
01
> m
•H v£>
3 0)
O J->
l-i -rl
•U ,£>
M
c rt
•H ,n
O
-H (3
cfl (U
JJ ,C
•H p.
J2 I
H O
C
a
O
B
cd
CO
4J
C
tu
to
0)
i-i
O
C
CU
XI ,
P.
•
M-l
O
!>,
0) H
CO i-l
j-i n)
d O>
O pi
O O
4->
>
H -H
0) T3
rC 0
-U 'H
O
CN
60
•H
56
-------
TABLE 13. EFFECT OF POTENTIAL INDUCING AGENTS UPON
BENZO[a]PYRENE HYDROXYLATION IN CONTROL TROUT HEPATIC
MICROSOMES IN VITRO
Concentration of arylhydrocarbon
(benzo[a]pyrene) hydroxylase
Compound added3 pAf
Noneb
Aroclor 1242 10
100
500
gNF 10
100
500
PB 10
100
500
nmol/min/mg
0.026 (100)c
0.025 (96)
0.026 (100)
0.022 (85)
0.005 (19)
0.004 (15)
0.003 (11)
0.027 (104)
0.023 (88)
0.021 (81)
aCompounds were added to incubation dissolved in 5 pliter N, W^dimethyl
formamide.
5 y liter N3 #-dimethylformamide was added to incubation.
cValues in parentheses are percentage of remaining activity.
TABLE 14. TYPICAL HEPATIC MICROSOMAL MONOOXYGENASE
ACTIVITIES OF RAT AND MOUSE FOUND IN AUTHORS' LABORATORY
Species and sex
Rat, male
Rat , female
Swiss mouse, male
Pre treatment
None
3-MC
None
3-MC
None
3^1C
ECODa
ERODb
nmol/min/mgc
0.72
13.2
0.31
5.65
2.00
4.61
0.38
64.8
0.80
39.9
0.51
6.82
Cyt P-450,
nmol/mg
0.71
1.54
0.67
1.07
0.54
1.05
Ethoxycoumarin-0-deethylation.
Ethoxyresoruf in-<9 -dee thylation.
cThese values represent specific activities (not V „ ), however these
nici x
measurements were made at substrate concentrations which gave at least 90%
of the V , value.
57
-------
This study has demonstrated differences in the spectral hepatic
hemoprotein properties of rainbow trout when compared to that of lake trout
(Salmo trutta lacustris) (Ahokas et al. 1976, 1977). Ahokas et al. have
indicated that the \ carboxyferrohemoprotein P-450 of lake trout is at
450.6 nm, whereas in tnis study the X of rainbow trout was consistently
at 449 nm. Furthermore, Ahokas et al. could not elicit type I substrate
binding spectra in lake trout using hexobarbital or PBO, but both of the
compounds elicited type I spectra with microsomes from rainbow trout. Type
II spectra found in this study were similar to those observed in rodent
species. Also, the trout microsomal hemoprotein P-450 was able to
metabolize methylenedioxyphenyl compounds resulting in the formation of
metabolic intermediate complexes seen frequently with mammalian hemoprotein
P-450 (Hodgson and Philpot 1974).
Several differences between the responses of rainbow trout and rodents
to certain xenobiotics are apparent from the present results.
Administration of Aroclor 1242 or phenobarbital to several mammalian species
results in an increase in the liver :body ratios and the amount of microsomal
protein obtained per unit of liver. However, this study has failed to
demonstrate such phenomena in rainbow trout with the same compounds.
The hemoprotein P-450 substrates were chosen to cover a broad spectrum
of monooxygenase activity. In many rodents, ethylmorphine-#-demethylation
is characteristic of cytochrome P-450 activity (inducible by
phenobarbitals), and benzo[a]pyrene hydroxylase and ethoxycoumarin-C'-
deethylation are catalyzed by both cytochrome P-450 and cytochrome Pi-450,
but 7-ethyoxyresorifin-O-deethylation apparently can be induced solely by
polycyclic aromatic hydrocarbon or mixed-type inducers.
3-Naphthoflavone is representative of a cytochrome P,-450 inducing
agent in rodents (Boobis et al. 1977). This study has demonstrated that it
is an effective inducer of enzyme activities associated with cytochrome
P,-450 in rainbow trout. However, only small qualitative alterations in the
nature of the constitutive hemoproteins were observed. For example, the Km
value for ethoxycoumarin-O^deethylation decreased, total hemoprotein P-450
content increased slightly, and the 455:430 ratio of the ethylisocyanide
spectra increased slightly. However, unlike the mammalian situation, no
blue shift in the Soret maximum of the CO complex of reduced hemoproteins P-
450 was observed. Similar observations were made using Aroclor 1242 as an
inducing agent.
As found by other investigations (Bend et al. 1973), this study
demonstrated phenobarbital to be totally devoid of inducing ability in
trout. This inability is probably not a problem of bioavailability since
the fish became sedated after injection with phenobarbital and ^ C-
phenobarbital-related material was found in the liver. Since the ratio of
phenobarbital to phenobarbital metabolites was not determined, the lack of
induction might be caused by a rapid metabolism of the barbiturate; however,
this possibility is unlikely considering the low levels of monooxygenase
activity. Furthermore, phenylbutazone and DDT, inducers of the barbiturate
class, are also incapable of inducing monooxygenation in fish. This study
has also demonstrated a lack of induction of the P-450 associated
58
-------
#-demethylation of ethylmorphine by Aroclor 1242; this lack contrasts with
the mammalian situation, in which Aroclor 1242 induces P -450 and P-450
associated activities.
A lack of induction of monooxygenation by phenobarbital has been
reported in the rat fetus (Guenthner and Mannering 1977). However, the
simultaneous administration of 3-naphthoflavone and phenobarbital resulted
in induction of cytochrome P-450 associated activities. A similar
experiment carried out in rainbow trout did not result in a synergism
(Elcombe and Lech, unpublished observations).
The use of a-Naphthoflavone and metyrapone to inhibit benzo[a]pyrene
hydroxylation by trout hepatic microsomes -in vitro resulted in some
interesting observations, a-Naphthoflavone strongly inhibited control and
induced AHH activity, but metyrapone had little effect. These results
contrast drastically with the mammalian situation, in which 3-naphthoflavone
stimulates control arylhydrocarbon (benzo[a]pyrene) hydroxylase (cytochrome
P-450) but inhibits cytochrome P,-450 mediated hydroxylation. lletyrapone
classically inhibits cytochrome P-450 (control and phenobarbital induced),
but has little effect on cytochrome P^-450. Hence, the results indicate
that the control hemoprotein P-450 of rainbow trout is similar to P,-450.
Other indications of this hypothesis are the relatively high control
activities of benzo[a]pyrene hydroxylase and ethoxyresorufin-0-deethylation
and a Soret band for carboxyferrohemoprotein P-450 at 449 nm.
However, the constitutive hemoprotein of trout hepatic microsome is not
cytochrome P,-450. This conclusion is clearly shown by several results:
The Soret peak of the carboxyferrocytochrome is at 449 nm, not 448 nm; the
EtNC 455:430 peak ratio is 0.28 not 1.0; and a comparison of apoprotein on
SDS-PAGE derived from trout and rat cytochrome P-450 demonstrated distinct
electrophoretic patterns.
This study also suggests that the physical properties of the induced
and control hemoprotein of trout hepatic microsomes are similar. For
example, although a 40% increase in total hemoprotein was observed after
pretreatment with 3"naphthoflavone, no shift in the Soret peak was observed;
furthermore, the ratio of the 455:430 peak of the ethyl isocyanide spectra
was altered only slightly after induction. Enzymatically, the induced and
control hemoproteins have similar qualitative but not quantitative
properties. Although induced and control hemoproteins showed similar
susceptibilities to inhibition by 6-naphthoflavone and metyrapone, increases
of 4,081%, 4,455%, and 1,178% in benzo[a]pyrene hydroxylation,
ethoxyresorufin-<9-deethylation, and ethoxycoumarin-0-deethylation,
respectively, cannot be explained on the basis of a mere 40% increase in the
constitutive hemoprotein content of the trout hepatic microsomes.
Qualitatively, the only significant alteration was the decrease in the Km
for ethoxycoumarin-<9-deethylation.
The results suggest that different subpopulations of hemoprotein(s) P-
450 exist in microsomes for control and 3-naphthoflavone treated trout.
This hypothesis is supported by the patterns of sodium dodecyl sulfate-
polyarylamide gel electropheresis in fish pretreated with 3-naphthoflavone
59
-------
(Figure 21, Figure 22). These patterns exhibited a novel band (which
included peroxidase activity) of an apparent molecular weight of 57,000.
This band was not observed in microsomes from control fish. The novel band
was relatively minor when compared to the total hemoprotein content, and
cannot account for the 40% increase in hemoproteins. Hence, g-
naphthoflavone and Aroclor 1242 apparently induce constitutive hemoproteins
in addition to the novel band at 57,000 daltons. Hence, the novel
hemoprotein induced by g-naphthoflavone and Aroclor 1242 apparently exhibits
extremely high monooxygenase activity toward benzo[a]pyrene,
ethoxyresorufin, and ethoxycoumarin.
STUDIES OF THE FATE OF ORGANIC POLLUTANTS IN FISH
Bolycyclic Aromatic Hydrocarbons; Naphthalene and 2-Methylnapthalene
Short-Term Exposures—
The tissue levels of C (yg naphthalene + metabolites/g) during an 8-h
exposure of fingerling rainbow trout to C-naphthalene (0.005 mg/liter) and
during a subsequent elimination period are shown in Figure 23 (Melancon and
Lech 1978).
Naphthalene was rapidly taken up by the fish, and the levels in the
tissues studied were from 22 to 340 times the initial water level of
naphthalene after 8 h of exposure. The average biliary concentration (bile
volume = 10 yliter) of naphthalene plus metabolites was 3.1 ug/ml (300 times
the initial water level of naphthalene) during an 8- to 32-h period.
Visceral fat had the highest level of C while blood and muscle had the
lowest of the tissues studied. The ^C levels in liver, gill, and whole
fish were between these extremes.
The results of an exposure at an initial water level of C-naphthalene
of 0.023 mg/liter are shown in Figure 24. The results of this exposure were
similar to those at the lower concentration of naphthalene in terms of the
relative levels of C in various tissues and in terms of tissue level at 8
h compared to initial water concentration. In this experiment the ratios of
tissue 1^C to water 1/^C after 8 h varied from 24 to 585. Bile levels (10 yl
of bile/fish) during the 8 to 32 h period averaged 8.2 yg/ml (360 times the
initial water level of naphthalene).
The half-lives for the elimination of C generally were less than 24 h
and were similar for the specific tissues at both exposure levels (Table
15). The exception was fat, for which the half-life was 31 h after the
lower-level exposure and 62 h after the higher-level exposure.
In order to evaluate the effect of multiple exposures on elimination,
fingerling rainbow trout were exposed to 0.005 mg/liter of *^C-naphthalene
for more than on 8-h period with 2-h elimination intervals. The elimination
curves on a whole fish basis for double and triple exposures are presented
in Figure 25. The half-life values for elimination were 12.3 and 9.8 h for
the double exposure and 15.5, 11.3, and 14.7 h for the triple exposure.
These values compare well with a half-life of 15.4 h for whole fish for
60
-------
A 8 C D
H
Figure 21. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of
microsomes from variously pretreated rainbow trout. Samples A,
D, and G; control microsomes at loadings of 45, 90, and 180 yg
of protein, respectively. Samples B, E, and H; 3-naphthoflavone
microsomes (100 mg/kg), loadings as above. Samples C, F, and
I; Aroclor 1242-induced microsomes (150 mg/kg), loadings as
above. Sample J; standards [Escherioh-ia ooli, RNA polymerase
(3, 6^, and 3' subunits), and bovine serum albumin]. Molecular
weights: Bands 1, 2, 3, 4, and 5 are 59,500, 57,000, 51,000,
48,000, and 45,000, respectively.
61
-------
Figure 22. Enlargement of the 40,000- to 60,000-dalton region of samples
D, E, and F from Figure 21. Molecular weights for bands 1-5
are as indicated in the legend to Figure 21.
62
-------
EXPOSURE ELIMINATION
UJ
3
CO
CO
£
cc
o
bo
ffi
Q_
UJ
UJ
<
I
bo
52
g
!<
UJ
O
o .003 h
o
.001
10 15 20 25
TIME (HOURS)
Figure 23. Tissue levels of lifC during exposure to 1 ^C-naphthalene (0.005
mg/liter) and subsequent elimination. Data are the average of
values from five trout except for the 8 h values which represent
10 trout. • = fat, *= whole fish, •= liver, a= gill,
o = muscle, a = blood, and + = exposure water.
63
-------
EXPOSURE ELIMINATION
LJ
10 15 20
TIME (HOUR)
25 30 35
Figure 24. Tissue levels of I'*C during exposure to 1'*C-naphthalene (0.023
mg/liter) and subsequent elimination. Data are the average of
values from five trout except for the 8 h values which represent
10 trout. •= fat, A= whole fish, •= liver, A= gill,
O = muscle, D = blood, and + = exposure water.
64
-------
UJ
Z)
CO
CO
o
60
a
CO
o
o
1.0
LJ
°- 03
Ijj w*°
z
LJ
0.1
0.03
1.0
0.3
1
0 10 20 30 40 50
TIME (HOUR)
Figure 25. Tissue levels of llfC during and after multiple exposures of trout
to 1^C-naphthalene (0.005 mg/liter). Each point represents the
average of values from five trout.
to
Solid lines indicate exposure
14C-naphthalene, and dashed lines indicate elimination periods.
D = two 8-h exposures, and o = three 8-h
• = one 8-h exposure,
exposures.
65
-------
TABLE 15. ELIMINATION HALF-LIVES OF 1I+C FROM FINGERLING RAINBOW
TROUT EXPOSED TO lltC-NAPHTHALENE IN WATER ON A SHORT-TERM BASIS
Exposure
level
mg/liter
0.005
0.023
Exposure
duration,
h
8
8
Elimination
duration,
h
24
24
Half -life of 1<+C elimination, h
Whole
Muscle Blood Liver Gill Fat fish
6.5 7.1 11.9 9.7 31.3 15.4
6.5 7.5 11.0 9.0 62.2 14.6
elimination of *C after a single exposure to 0.005 mg/liter of
naphthalene for 8 h and show no striking differences resulting from the
multiple exposures.
Long-Term Exposures—
Statis exposures such as those described above are of limited value
because the water concentration of the compounds under study decreased
during the exposure period. Therefore, utilizing a. continuous flow system,
we studied the uptake of C-naphthalene (0.023 mg/liter) by fingerling
rainbow trout.for approximately 4 weeks and the subsequent elimination of
accumulated C by the fish (Figure 26). As in the short term studies,
muscle and blood levels of C were similar and were considerably lower than
liver concentrations. Muscle and blood levels of naphthalene were 25 times
the water concentration of naphthalene and liver levels were 175 times the
water level of naphthalene after 16 days of exposure.
A similar experiment was performed to study the uptake and elimination
of l^C-methylnaphthalene (Figure 27). In this study, liver again contained
the highest levels of the C label, while muscle and blood contained lower
levels. The concentration of 2-methylnaphthalene in all three tissues had
reached a plateau (100 to 300 times the water concentration of
2-methylnaphthalene) long before the end of the exposure period. Muscle
levels of 2-methylnaphthalene reached a maximum after 9 days of exposure and
showed definite decreases during the remainder of the exposure. After the
fish had been in fresh water for 4 days, the radioactivity in 20 uliter of
blood was no longer significantly above background. The half-life values for
elimination of the two chemicals from the tissues studied are given in
Table 16.
The elimination of C accumulated from C-naphthalene during the
long-term exposure took much longer than after the short term exposures.
Although blood C levels showed an early decrease, all three tissues
studied showed a gradual elimination of C. The elimination of C
accumulated from C-2-methylnaphthalene during long-term exposure was quite
different from that following long-term exposure to C-naphthalene. After
66
-------
3nssu jo
3N3~IVHlHdVN 8r
ao
4-1
C
3
cr1
CU
CO
fi
3
CO
TJ
fi
cd
cu
c
cu
rH
r*
4-1
ft
cd
fi
1
o
J-
r-t
o
4-1
0)
^_l
3
CO
o
pi,
a
^*»
cd
i
CN
cd
60
C
.1
"Pi
M
j^
-a
4-1
3
o
J^
4J
C
•H
O
j-
i-t
MH
O
CO
rH
CU
^
cu
i — i
01
3
CO
CO
•H
H
1
^n
cd
o
•H
4-1
J_J
cu
^
CU
rfl
•U
*rt
G
cd
»*
4J
3
O
j_i
4J
a>;
•H
UH
a
0
f^
M-l
CO
01
rH
cd
£>
UH
O
CU
60
cd
rl
cu
^
cd
cu
4-1
CO
4-J
c
0)
CO
cu
J_j
ft
cu
j_i
4-1
C
•rl
O
ft
pfi
O
cd
w
•
C
0
•H
4-1
cd
C
•H
a
•rl
rH
CU
J_,
CU
4-1
cd
J5
c
•rl
CU
G
CU
rH
cd
f*
4-1
r*
ft
cd
G
1
o
*
-H
14_l
o
fi
0
•H
4-1
cd
f^
4-1
fi
cu
o
c
o
o
cu
60
cd
Q)
^
cd
cu
t~j
f-i
•
o
J_1
OJ
i-rj
^4
cd
fi
cd
4-1
CO
01
,£J
4-1
40
fi
Ol
CO
01
^_j
ft
0)
^1
CO
cu
fi
•H
H
cu
3
CO
o
ft
X
cu
II
"H
TJ
fi
cd
•s
13
o
o
T_l
c\
II
a
A
CU
rH
a
CO
3
a
n
o
f,
^
cu
^
•rl
rH
II
^
•
^
cu
4-1
•rl
1
i~n
60
a
ro
CM
O
•
o
CO
cd
CU
t-l
3
CO
0
ft
cu
01
X!
4-1
60
G
•rl
rl
3
13
•
^
0)
4-1
cd
CM
0)
60
67
-------
QJ
,e I
4J H
fi
cfl
QJ
c
QJ
T3 ,C QJ
C 4-1 H
c3 0) O
& co
• I 3
4-> CN a
3 I
O U II
V-i J-
w- o
cx >
cfl -H
£8
ij t^
QJ MH
f QJ
CN 3
QJ 0)
s-i >
3 nj
ca
o •> -I-1
n) C
TJ 0)
co
0)
CM t-i
I
rt OJ
o -
M
C OJ
O >
-H -H
a)
a) w
60 -H
0) rH
M *•-.
a) oo
> g
03
r-
0) rH
J3 O
H •
o
• en
M Cfl
o 3
M
>-i QJ
ai M
C -U
•r) fi
M -rl
3 O
13 PH
T) CO
O
- ,
3 O 01
tfl
W
c
o
-rl
4J
MH -H
o e
•rl
CO rH
rH QJ
QJ
> 4-1
QJ C
rH QJ
0) U*
3 QJ
CO CO
CO rQ
•H 3
H CO
ft M
CJ2
ca
QJ s
4J OJ
rl
. 60 3
4-1 a w
•H O
4-1 rl ft
C 3 X
0) T3 QJ
CO
QJ rl II
)H 0)
ft 4-1 +
QJ t«
rl & T3
CD c co
QJ -H
c
•H QJ 13
rH CI O
QJ O
rH H rH
tO tO rd
a js
QJ n) „
> c a
CM
QJ
rl
3
bO
•1-1
68
-------
TABLE 16. ELIMINATION HALF-LIVES OF lkC FROM FISH EXPOSED TO
AQUEOUS ll*C-NAPHTHALENE OR 14C-2-METHYLNAPHTHALENE FOR SEVERAL WEEKS
Half-lives, h
Exposure Muscle Liver Blood Whole fish
Trout 909 343 379
Naphthalene
0.017 mg/liter
Trout 13a 211 23
2-Methylnaphthalene 711
0.023 mg/liter
Carp 31b 59b
2-Methylnaphthalene 1942 781
0.013 mg/liter
Bluegill sunfish < 24C
2-Methylnaphthalene 353
0.013 mg/liter
aWhen two values are given, the upper value is the early rapid phase of
elimination and the lower value is the latter slower phase. The slope and
intercept for the slow phase of elimination were calculated using the data
from days 4-36. The data from days 0-2 were corrected for this, and the
resulting values were used to calculate slope for the rapid phase of
elimination from days 0-2.
Data for flow phase, days 8-73, and for rapid phase, days 0-3.
GData for slow phase, days 1-26, about 60% of * C eliminated during first
day.
exposure to C-2-methylnaphthalene, C levels in blood dropped rapidly and
an initial rapid drop in ^ C levels in muscle was observed followed by a
gradual loss comparable to that observed with naphthalene. Because of the
fluctuations of the C levels in liver, the elimination from liver was
calculated as a single-phase elimination and resulted in a half-life similar
to that observed with naphthalene.
Levels of C in bile increased during the early part of the exposure,
reaching a maximum at 2-3 weeks for each compound. After this period, C
reaching a maximum at 2-3 weeks for each compound. After this period, C
levels in bile dropped more rapidly in trout exposed to 1^C-naphthalene than
in those exposed to C-2-methylnaphthalene. At 24 h naphthalene and
metabolites in bile totaled 12 jag/ml (approximately 500 times the water
concentration of naphthalene), and 2-methylnaphthalene and metabolites
totaled 28 ug/ml (approximately 1,600 times the water concentration of
69
-------
2-methylnaphthalene). The maximum for naphthalene and metabolites was 300
yg/ml (approximately 13,000 times the water concentration of naphthalene)
while that for 2-methylnaphthalene and metabolites was 400 yg/ml
(approximately 23,500 times the water concentration of 2-methylnaphthalene).
Initially greater concentrations of ^C in bile resulting from exposure to
C-2-methylnaphthalene compared with exposure to C-naphthalene were
probably related to the greater uptake of 2-methylnaphthalene. Later in the
exposure and during the first week of elimination, however, the differences
were much more striking and may be related to the considerable loss of C
from muscle of trout exposed to ^C-2-methylnaphahtlene at these times.
Small carp and bluegill sunfish were exposed similarly to C-2-
methylnaphthalene (Table 17 and Table 18) (Melancon and Lech, 1979).
Because of the very small size of the latter, C levels were determined
only for the whole fish. The elimination of C from these species was
similar to that observed with trout. A substantial loss of C from tissue
occurred during the first 24 h of elimination followed by a much more
gradual loss. The half-lives for elimination of C from the various
tissues in these experiments are listed in Table 16.
While biliary C in the trout reached a maximum of 10,000 to 20,000
times the water level of C with both compounds, biliary C in the carp
was over 100,000 times the water level of 9/on several occasions.
Interestingly, these high levels of biliary C continued well into the
elimination period after exposure of trout and carp to C-2-
methylnaphthalene. The levels of biliary C were much however, during the
elimination period following exposure of trout to C-naphthalene.
The biliary levels of C in carp exposed to C-2-methylnaphthalene
were particularly interesting. We have suggested (Statham et al. 1976) the
use of fish bile in the monitoring of aquatic pollution. The results
support this suggestion since bile contained C at over 100,000 times the
level of C in the exposure water and these concentrations persisted for
several days after the exposure was terminated.
Metabolism Studies—
The •*• C present in bile and in acetone extracts of muscle and liver
from trout exposed to 0.5 mg/liter of C-naphthalene and **C-2-
methylnaphthalene was examined for the presence of metabolites (polar *^C-
labeled material) by thin-layer chromatography (Figure 28). With CCl^ as
the solvent, the unchanged hydrocarbons had Rf-values of approximately 0.5,
while metabolites remained at the origin. Because of the volatility of the
unchanged hydrocarbons, much of the **C was lost during TLC, but the percent
of the spotted amount of C which remained at the origin gave some degree
of quantification of the amount of metabolites present. For both
hydrocarbons bile contained at least 65-70% metabolites, liver 5-10%
metabolites, and muscle < 1% metabolites. More recent values of percent of
metabolites in bile derived from hexane:water partitioning are generally
over 85% and frequently over 95%.
70
-------
LJ
C\J
UJ
_o
-00
.
CD
10 cr
O
O
cc
-CM
O
H3AH
310SDIAI
I
ft
I
u a
a) cu
4-1 60
X cu
cu ca
I
a>
ft
cu
M
j- CU 0
-30
CO I
H CO rH
CU -rl
•H a
rH CU CO
•^ £3 CU
60 O
g 4-1 cJ
Ol -rl
in o
• CO 4-1
o a
m cu
o o co
4-1 CU
T3 O
cu
co a)
O rH
ft -rl . .
X & >
CU -H
U-l 4-1
4-1 O O
3 CO
O CO O
K CU -rl
4J rH T3
s &2
5 I
rO CO Q)
C 43
•H H
CU
31
a a
J-l
•H T3
o cu
rH
cu a
43 M
H -H
a
. cu
CU 43
4J 4-1
cO
4-J O
C
Cfl
e
O
4-1
•H
>
CU
co
•H CU CO
4-1 rH
O CO J-
O 4-1 U
•H 43 O
*"O PJ
CO 4-1 T3
CO CU CU
•H 0 43
4J I ft
CM CO
O O 60
J- O
CO- 4-1
0) CO
•rl O O
M-l >-!
O CU 43
H (3 O
ft CU
rH CU
U CO H
hJ 43 CU
H 4J &
H T)
CU 3 •
43 O T)
4-1 ft C
6 3
O O O
4J O ft
'rj 4J O
cu c a
•rl CU
rH H TJ
ft CO CU
ft ft 4J
CO cO
CU rH
4-1 CO X
•rl -rl O
•H ft T3
4-1 O >i
CO ft O
O ft B
•H cfl O
CO CU
t-t 43 OJ
rH
cO
J-J
CO
O >-i
O ft
CO
CU
4J O
<4H -rl ft
O rH ft
•H
gs? 4^
CO 0
cO
cy
13
cu
ca
CO Cfl 4-1
(U 4-1 CO
M a 43
ft CU 4J
X ca
cu cu co
J-J 4J
ft B
ca cu cu
•H l-l CO
00
CN
00
•H
71
-------
TABLE 17. UPTAKE AND ELIMINATION OF lltC-METHYLNAPHTHALENE BY
CARP AFTER EXPOSURE TO 0.013 MG ll|C-2-METHYLNAPHTHALENE/LITER
Time,
days
Exposure 1
4
8
15
22
26
Elimination 1
2
3
8
19
26
32
73
ll+C-2-methylnaphthalene and metabolites
yg/g of tissue
Muscle
0.67 ± 0.02
1.47 ± 0.28
1.31 ± 0.09
1.42 ± 0.20
1.13 ± 0.08
1.60 ± 0.17
0.96 ± 0.08
1.01 ± 0.08
0.62 ± 0.14
0.44 ± 0.07
0.26 ± 0.04
0.41 ± 0.09
0.45 ± 0.11
0.22 ± 0.08
Liver
5.82 ± 0.75
7.55 ± 1.06
6.89 ± 0.76
8.31 ± 0.82
10.19 ± 0.49
12.82 ± 1.73
7.20 ± 1.03
5.81 ± 0.48
6.13 ± 1.17
2.76 ± 0.45
1.56 ± 0.63
2.05 ± 0.42
1.88 ± 0.29
0.63 ± 0.09
TABLE 18. UPTAKE AND ELIMINATION OF 11+C-2-METHYLNAPHTHALENE BY
BLUEGILL SUNFISH AFTER EXPOSURE TO 0.013 MG
1 ** C-2 -METHYLNAPHTHALENE/ LITER
C-2-methylnaphthalene and metabolites
Time, yg/g of tissues
Exposure
Elimination
days
1
4
8
15
22
26
1
2
3
8
19
26
Whole fish
2.17 ± 0.35
2.94 -t 1.14
5.41 ± 1.65
3.68 ± 0.83
6.30 ± 2.29
5.24 ± 1.36
2.16 ± 0.29
2.25 ± 0.52
1.62 ± 0.26
1.86 ± 0.41
1.14 ± 0.40
0.58 ± 0.09
72
-------
Additional TLC in other solvents demonstrated that most of the
metabolites in bile were more polar than the hydroxyl derivatives of
naphthalene and 2-methylnaphthalene and were probably present as
conjugates. Investigations are continuing on the isolation and
identification of these metabolites, and the results of these studies will
be reported at a later time.
The data for the elimination of ^C-naphthalene following short-term
and long-term exposures indicated that elimination of C after the
long-term exposure was much slower than after short-term exposures.
Although no information of short-term exposure and elimination using C-2-
methylnaphthalene has been presented, some limited data indicate that the
half-lives for elimination of this compound from several tissues following
8-h exposures were considerably < 24 h. Elimination of C from muscle and
blood after long-term exposure to C-2-methylnaphthalene had a rapid phase
with half-lives not greatly different from those following short-term
exposure to C-naphthalene or C-2-methylnaphthalene. A much slower
elimination phase also occurred, which was comparable to the elimination
seen following long-term exposure to C-naphthalene.
14
A possible explanation for the slower elimination of C following
long-term exposure to these compounds as compared to that following short-
term exposures was suggested by two recent reports. Roubal et al. (1977)
found that metabolites of naphthalene are present in tissues of coho salmon
fingerlings following intraperitoneal injection of ^C-naphthalene, and
Sanborn and Malins (1977) reported that naphthalene is released more rapidly
from shrimp larvae than are naphthalene metabolites.
Although in this study TLC of the muscle extracts from trout exposed to
0.5 mg/liter ^C-naphthalene or ^C-2-methylnaphthalene for 24 h showed that
< 1% of the l^C was present as metabolites, the possibility existed that the
longer term continuous-flow studies at lower levels might have resulted in
greater percentages of metabolites in muscle. Despite the relatively low
levels of -^C occurring in muscle samples at certain stages of the long term
study, estimation of the percent of metabolites present in muscle seemed
important. Therefore^ muscle filets from fingerling rainbow trout, which
had been exposed to C-naphthalene or ^C-2-methylnaphthalene for either 1
day, 4 weeks, or 4 weeks with a subsequent elimination period, were
extracted, and the C present in these extracts was examined by TLC. The
data in Table 19 show that the proportions of ^C in muscle present as polar
material were greater after a week of elimination than during the exposure
period for both compounds. In the case of 2-methylnaphthalene. the
difference in percent of metabolites is striking. Only 1% of C in muscle
was present as metabolites during the exposure and at the beginning of the
elimination period; however, 24% of C in muscle was present as metabolites
during the slow phase of elimination. No striking change occurred in the
percent of metabolites present in the muscle from fish exposed to C-
naphthalene.
Following long-term exposure of trout to C-2-methylnaphthalene the
elimination of C from muscle initially was very rapid at a time when the
l^C was present mostly as 2-methylnaphthalene. Later, the elimination was
73
-------
slower when a substantial fraction of the * C represented metabolites of
2-methylnaphthalene. These results are consistent with differential rates
of elimination of 2-methylnaphthalene and its metabolites from muscle.
During exposure of trout to naphthalene a significant fraction of the C in
muscle was present as metabolites early in the exposure period. Although
this fraction increased after a week of elimination, the elimination rate of
C was monophasic at a rate similar to the slow phase of elimination of C
from the muscle of trout exposed to 2-methylnaphthalene.
TABLE 19. FRACTION OF 14C IN MUSCLE FROM TROUT
EXPOSED TO 14C-NAPHTHALENE OR 14C-2-METHYLNAPHTHALENE
PRESENT AS POLAR COMPOUNDS
Exposure
14 a
C as polar compounds,
Naphthalene
1-day exposure 21
27-day exposure 12
27-day exposure plus
9-day elimination 34
2-Methylnaphthalene
1-day exposure 1.5
26-day exposure 1.1
26^day exposure plus
7-day elimination 24
aValues shown are derived from muscle samples analyzed at
the end of the indicated experimental periods.
Whether these metabolites are formed in muscle or in another tissue
such as liver is not known. During long-term exposures bile was found to
contain C at thousands of times the water exposure levels. However, these
biliary metabolites were not necessarily excreted in the feces and the
possibility exists of enterohepatic circulation with subsequent accumulation
of these metabolites in such tissues as muscle. Regardless of how these
metabolites accumulate in muscle, the possibility of such accumulation
during relatively long-term exposures of fish to chemicals must be
considered when experiments are designed to evaluate such factors as
bioaccumulation and equilibrium concentrations of various chemicals in fish.
Compared to naphthalene, 2-methylnapthalene is taken up more rapidly by
rainbow trout, intitally is accumulated to higher levels in all tissues
studied, is stored in muscle to a much greater extent as the parent compound
74
-------
and is eliminated more rapidly from the muscle, liver and blood. Data also
suggest that over longer periods of time, exposure of trout to naphthalene
might lead to higher tissue leels of the parent compound plus metabolites
than would be the case with 2-methylnaphthalene. Although long-term
exposures are often difficult to interpret because of possible enterohepatic
cycling of metabolites within the fish or cycling between fish, water, and
flora, the results reported herein indicate that short-term exposures alone
could be misleading for predicting long-term tissue levels of certain
chemicals .
A summary of the biliary levels of C resulting from the exposure of
trout to C-naphthalene and trout, carp, and sheepshead to C-2-
methylnaphthalene is presented in Table 20. In general, exposure to C-2-
methylnaphthalene resulted in higher biliary levels of C than did exposure
to C naphthalene. Biliary levels of C in fish exposed to C-2-
methylnaphthalene were higher for carp than for trout or sheepshead.
The differences in biliary levels of C in trout exposed to these
chemicals may be explained in part by the greater tissue levels of C
resuting from exposure to l^C-2-methylnaphthalene. Biliary levels of *^C
during the elimination periods following 4 weeks of exposure to these
chemicals did not substantiate this explanation. During the elimination
periods the biliary levels of ^C in fish exposed to * C-2-tnethylnaphthalene
were much higher than those in fish exposed to C-naphthalene at times when
tissue levels of C in the methylnaphthalene-exposed trout had dropped
below those of the napththalene-exposed trout.
When quantities of bile from the long-term exposures of trout to C-
naphthalene or 2-methylnapthalene were great enough, pooled bile from each
sampling time was examined by hexane :water partitioning. In general, over
98% of the 14C in the bile represented metabolites, and over 90% of the 14C
represented very polar metabolites, probably conjugated metabolites.
The TLC of bile from small carp exposed to C-2-methylnaphthalene
showed that the peaks of C were much more polar than 2-methylnaphthalene
and 2-methyl-l-naphthol (Figure 29). The standards, naphthalene sulfate,
naphthalene glucopyranoside, and naphthalene glucoronide did not co-
chromatograph with either of the two major radioactive areas resulting from
TLC in the butanol :NH^OH:water solvent system. TLC of 'the biliary C from
similarly exposed larger carp with the same solvent system showed a similar
distribution of radioactivity (Figure 30). While biliary 14C from
sheepshead exposed to C-2-methylnaphthalene also resulted in two
radioactivity peaks in this solvent system, the peaks different from those
present in carp. As shown later, biliary C in trout appears to be
intermediate between these two species (Figure 31).
Chlorinated Hydrocarbons
The uptake, metabolism, disposition, and elimination of several
chlorinated benzenes also were studied. The first two, pentachlorophenol
(PGP) and pentachloroanisole (PCA), which differ only by a methyl group,
were studied at the same time and are discussed together. The third —
75
-------
O
EH
X
en
M W
Fr. ly
MH f-*
T W
b H
5*
U H
gs
w * w
« H
^C '^C
H *T*
i-J H
S£
r^
rH
U
03
3
tH
>%
(-1
tO
•rH
H
•H
m
T3
>. 1 fi
h 1 4-1 3
tO H fi O
•H Q) CU CX
•H > ^i 0
. i MI f-t **
•H CU CO O
M rH CX CJ
rH
Cfl
4J
•H
rH
cu
00
CO
(-1
0)
o>
rH
•§
CO
M
01
4-1
§
43
CO
•rH
4-1
M rH
01 01
4-1 >
Cfl 01
S H
A
CO
01
4-1
•H rH
rH B
O "--
43 00
ert f3
CO t3
4-1
CU
e
cu C
£ o
3 -H
CO 4J
O tfl
CX r-l
83
M
M cu
rH 4J
Ol 'H
> ^H
CU %^^-
rH 00
a
^
4J
43
00
•H 00
01
£
co
cu
. -j
*rt
o
01
CX
CO
13
C
o
CX
e
o
CJ
O sj-
r-^ oo
CO CO
CM CN
0 0
o o
O 0
CO
43 43
-3- 00
CN
m m
0 0
o o
0 0
rH VO
I-l
4-* 4J
3 3
o o
££
0)
fi
CU
i-H
cfl
4J
43
CX
CO
Z
rH O
O> o
in CM
A
ON
rH
-3- 1^
rH CN
O CO
0 0
CO
>~.
tO to
43 "0
OO vO
i-H
co r^
»
CO
43 -d 43 43
-3- sT «* -3"
CM rH CM CM
m ro ~3- o
o CM cy> co
o o ** -3-
o o o o
i-H -3- -3- CM
i-H c^ o\
Jl , 1 11 I 1
+J +J +* HI—'
3333
O O O O
fH^EHEH
cu
cu
rH
rrt
CD
43
4-1
43
O.
CO
fi
i-H
5*.
4-1
CU
P
CN
O O O
O O- vO
CM O\ -3-
A M A
i-H i-H rH
^3-
rH
in-3" rH
co m cr>
CO vO vO
i-l O O
CO
>>
cfl
•0 43 43
00 >3- »3-
CN CN
co r^ co
rH co r-»
O co -3-
O o o
-* m o
CM m
O
M
rH
P. CX CX
M r< r<
c3<3c5
i-H
vO
CO
vO
CM
O
O
43
oo
•3-
CN
r-~
o
CD
O
m
m
I
o
m
-3-
"O
cfl
0)
43
CO
0)
(U
&
•
M
cu
4->
cfl
>
43
CO
0)
M
<*H
G
•H
f-i
4-1
~3-
CM
CO
p*4
p
rH
CX
43
00
<4H
O
cu
I-l
3
Cfl
o
PL*
&
CO
76
-------
3000
5
£ 2000
UJ
a.
1 1000
Q
I
UJ
m 3000
i
-h 2000
1000
1000
15
CM FROM ORIGIN
Figure 29. Thin-layer chromatography of biliary 1'*C from carp exposed to
0.337 mg of 11*C-2-methylnaphthalene/liter for 24 h. The solvents
used were A = CCL , B = benzene:acetone (5:1) and C = the
organic phase of butanol:NHi(OH:water (4:1:5). The circled
numbers represent the mobility of standards where 1 = 2-methyl-
naphthalene, 2 = 2-methyl-l-naphthol, 3 = 1-naphthylsulfate,
4 = 1-naphthylglucopyranoside and 5 = 1-naphthylglucuronide.
77
-------
20001
SHEEPSHEAD
CM FROM ORIGIN
Figure 30. TLC of biliary 1>tC from carp and sheepshead exposed to ltfC-2-
methylnaphthalene. The carp were exposed to an initial level of
0.47 mg/liter for 24 h, and the sheepshead were exposed to an
initial level of 0.072 mg/liter for 48 h. The solvent used was
the organic phase of butanol:NH OH:water (4:1:5). The circled
numbers represent the mobility of standards 1 = 2-methylnaph-
thalene, 2 = 2-methyl-l-naphthol, 3 = 1-naphthylsulfate, 4 =
1-naphthylglucopyranodide and 5 = 1-naphthylglucuronide.
78
-------
18
i —
bo
•
bo
_ Pentachlorophenol
Pentach loroan i sole
2 4 6 8 10 12 14 16 18 24
hours
Figure 31. Time course of PCP and PCA in several tissues of rainbow trout.
Data are calculated as micrograms of PCP or PCA per gram wet
weight. Each point represents the mean ± S.E. from at least six
fish in two separate uptake studies.
79
-------
trichlorobenzene—is discussed separately.
Bentachlorophenol and Bentachloroanisole—
Small rainbow trout were exposed to ^C-PCP or l^C-PCA as described in
the methodology section.
The data shown in Figure 31 illustrate that both PGP (upper panel) and
PCA (lower panel) are taken up rapidly from water by rainbow trout. Since
these static exposures were intended to determine the time course of uptake
prior to elimination studies, the plateau of the tissue concentrations
should not be taken as steady tissue levels. The plateau in the tissues
shown is more likely because of a rapid removal of PGP and PCA from the
exposure water rather than a true steady state due to saturation of
tissues. Clearly, C from PCA was concentrated rapidly in adipose tissue,
reaching a tissue C to initial water C ratio of approximatley 4,000
while the ratio for PCP was approximately 400. On the other hand, PGP
appeared to reach higher concentrations in liver than PCA.
Elimination of C from tissues of PCA- and PCP-exposed rainbow trout
is shown in Figure 32. Although some redistribution of PCP in the muscle
and fat during the washout period occurred, the time course of elimination
clearly shows that PCA is retained for a much longer period of time in
rainbow trout than is PCP. The half-lives for PCP and PCA calculated from
these curves are shown in Table 21, and the magnitude of the difference in
retention of the compounds is reflected in the extremes in the half-lives
for PCP (hours) when compared to PCA (days).
TABLE 21. HALF-LIFE OF PCP AND PCA IN RAINBOW TROUT TISSUES3
Half-lives
Chemical Blood LiverFatMuscleGillsHeart
PCP
PCA
6.2 h
6.3 d
9.8 h
6.9 d
23.7 h
23.4 d
6.9 h 10.3 h
6.3 d
6.9 h
lData calculated from elimination studies shown in Figure 30.
Figure 33 shows the concentrations of C, calculated as PCA or PCP, in
water, bile, blood, and fat of trout exposed to ^C-PCP (upper) and * C-PCA
(lower). At both sampling times shown, the PCP concentration in bile was
much higher than that in fat in the PCP-exposed trout, while the opposite
was true with the PCA exposure. The water concentration shown is for
reference only and is the value from samples taken at zero time.
80
-------
l_
o
E
no
*
bo
8
7
6
5
4
3
2
1-
80. r-
(I—
60-
40
20
Pentach loropheno I
Pentachloroanisole
o 0.5 i
3
days
Figure 32. Elimination of lkC from PGP- and PCA-exposed rainbow trout.
After a loading exposure the fish were transferred to fresh
running water and sampled at the indicated times. Note the
difference between time scales in the upper and lower panels,
Each part represents the mean ± S.E. from six fish in two
separate experiments.
81
-------
fe
rE
•
&o
300
200
100
6
5
4
3
2
60
40
20
10
8
6
4
2
_
-
-
-
—
-
H;0
-
-
-
-
-
_
—
H2°
Pentachlorophenol
ji-
T
1
blood
bile
blood
f_
bile
T
1
fat
T
1
-. -1
—
fat
••
T
1
blood
T
1
~. — •
—
•• "^
bile
T
1
fat
Pentachloroanisole
^ -^
fbi^d
T
1
bile
"
fat
4hr
8hr
exposure time
Figure 33. lkC in blood, bile, and fat of rainbow trout exposed to llfC PCP
and1'*C PCA for 4 and 8 h. The height of the bars represents
the mean ± S.E. from at least six fish in two separate exposures.
The water concentration is for zero time and is presented as a
reference point only.
82
-------
Thin-layer chromatographic analysis of acetone extracts of the muscle,
liver, and bile from PGP-exposed trout is shown in Figure 34. The data
clearly indicate the absence of detectable PGA in any of the tissue samples
analyzed from PCP-exposed fish. The polar material seen at the origin in the
bile and liver appear to be PCP-glucuronide, since treatment of the bile
with 3-glucuronidase decreased this peak and increased the radioactivity in
the area corresponding to the authentic PGP standard. A third radioactive
peak can be seen with an R^-value slightly less than that of PCP, but this
material was not identified. The results of gas chromatography/mass
spectroscopy (GC/MS) analysis of partially purified (by TLC) extracts of the
muscle, liver, and hydrolyzed bile from PCP-exposed trout are shown Table
22. Before chemical methylation of the extracts, the only PGP-related
compound found was unchanged PCP. After methylation the latter material
afforded the identical retention time and MS of PCA. No GC/MS evidence
existed for PCA, tetrachlorohydroquinone, or tetrachloroquinone in the
tissues examined. Although similar studies of these tissues from the PCA-
exposed trout revealed only PCA in muscle and a trace of more polar material
in liver (Figure 35), bile appeared to contain a polar metabolite of PCA.
Treatment of the bile with g-glucuronidase (lower panel) resulted in the
appearance of a new radioactive peak corresponding to the mobility of the
PCP standard.
Since the appearance of conjugated PCP in the bile of PCA-exposed trout
suggested demethylation of PCA, the effect of PBO—an inhibitor of
microsomal mixed-function oxidases—on the biliary metabolite pattern of
TABLE 22. THE GC/MS AND TLC ANALYSIS OF TISSUE EXTRACTS
FROM RAINBOW TROUT EXPOSED TO
Sample
Molecular ion (M+)
observed
14C-Rf value
PCP standard
PCA standard
Liver extract
Methylated liver extract
Muscle extract
Methylated muscle extract
Bile extractb
Methylated bile extract
264, 266, 268, 270
278, 280, 282, 284
264, 266, 268, 270
278, 280, 282, 284
264, 266, 268, 270
278, 280, 282, 284
264, 266, 268, 270
278, 280, 282, 284
0.46
0.85
0.46
0.85
0.46
0.85
0.46
0.85
Glickman et al. (1977) for experimental details.
Hydrolysis by $-glucuronidase caused residue at R^ of
an R of 0.46.
zero to migrate to
83
-------
Pentachlorophenol Exposure
o
o
01 2 3 4 5 6 7 8 9 10 11 12 J3 14
cm from origin
Figure 34. Thin-layer radiochromatogram of samples prepared from tissues of
rainbow trout exposed to ^C-PCP. Clear spot is authentic PGP;
hatched spot is authentic PCA.
84
-------
Pentachloroanisde Exposure
CVI
'o
CD
Bi le t p gl ucuronidase
cm from origin
Figure 35, Thin layer-radiochromatogram of samples prepared from tissues of
rainbow trout exposed to 1IfC-PCA. Clear spot is authentic PCP;
hatched spot is authentic PCA.
85
-------
PCA-exposed trout was studied. The data in Table 23 show that inclusion of
1 mg/liter of PBO in the tank water reduced the bile content of C.
Calculation of the distribution of radioactivity in bile—based on the TLC
analysis of the bile from the control and PBO groups—indicated that the
concentration of PCP-glucuronide was reduced in bile in the PBO-treated
fish, and the concentration of unconjugated-PCA was increased (Table 23).
The results of GC/MS analysis of the radioactive peaks extracted from the
chromatogram shown in Figure 35 are presented in Table 24. The GC/MS data
substantiate that the compound in the bile (Rf = 0.46) of PCA-exposed trout
was PC P.
The studies with PGP and PCA indicate that they are taken up rapidly
from water by rainbow trout and assimilated into various tissues. Although
the amounts of C in the tissues of trout exposed to C-PCP and PCA cannot
be taken as true steady-state tissue concentrations, these short-term
exposures indicate that the concentration of PCA in fat was much higher than
that of PGP. The data in the elimination studies agree with this finding,
and the high retention time of ^C from PCA (half-life in days) probably
reflects the high lipid solubility of PCA as compared with PGP. On the
other hand the concentration of C derived from PGP is much higher in the
bile—both in the uptake studies and the elimination studies—than the C
derived from PCA. We have reported previously that phenols, such as Bayer
73 and TFM, can be conjugated and exposed in bile in high concentrations in
rainbow trout (Statham and Lech 1975). This latter observation probably
accounts for the high concentration of C derived from PCP that is found in
rainbow trout bile in this study.
The identification of conjugated PCP in bile of rainbow trout exposed
to C-PCA indicates that rainbow trout are capable of demethylating PCA in
vivo. This finding is supported further by the capability of PBO—an
inhibitor of mixed-function oxidases—to decrease the amount of PCP
glucuronide in bile while concomitantly increasing the amount of unchanged
PCA; the finding is also in agreement with studies which have confirmed the
dealkylation of several foreign compounds in fish in vivo (Hansen et al.
1972, Olson et al. 1977). Although the difference in magnitude of biliary
excretion of C derived from PCA as opposed to PCP is difficult to explain,
several reasons are plausible. PCP is a phenol, which can be conjugated
directly with glucuronic acid and excreted in bile, while PCA must first be
demethylated before it is conjugated with glucuronic acid, and demethylation
may be the overall rate-limiting step in biliary excretion. The high
concentration in tissues of ^C from PCA as opposed to that of PCP also must
be considered. The transfer of PCA from its tissue depots to sites of
metabolism and excretion (gills, liver, kidney) may well be the overall
limiting factor in elimination of PCA.
1,2,4-Trichlorobenzene—
Small rainbow trout were exposed to C-l,2,4-trichlorobenzene (TCB) in
a static system or in a continuous flow delivery system in a similar manner
to the exposures previously described for C-naphthalene and 2-
methylnaphthalene. The results of the short-term static exposure are
presented in Table 25, while those for long-term exposure are presented in
86
-------
PK
0
w
tJ
M
P3
S3
M
rU S
-i I
o
O -i
S3 0
O H
M
H Q
ED w
PP CO
H p
Pi K
H X
co w
M
n H
S3 O
0 *
o
« r*
PH O
CO
fo s
O M
O O
• •
0 0
oo r»
/— N
M
CU
4-1
*|-(
rH
^^,
rH 60
o e
VJ
I i ,__)
fj O v-/
O PQ-
O PM
C
tf{
T1
r^ ~"
QJ f\
O _j
cfl ^
IxTl
Cj
5 cu
J_(
j3 CU
S Q
^ Q S
CU 4-1 " 1-4 M X
CU -H rH ~,
3 Cl rH
CO
EC
CJ
1
s~*
-f-
s
^
C
C TD
o cu
•H >
M CU
CO W
rH 43
3 0
O
cu
rH
CU
rH
0,
S
J§
VD vO vC
CN4 CS csj
** *» A
|"S», fN^ (»>„
VO ^O ^O
CM CM CM
m in in
^D vO ^O
CM CN CM
CO CO CO
^O vO ^O
CM CM CM
0*^0 "^ *^" *^
r^ oo r~> oo oo oo
CM CM CM CM CM CM
oo CM* oo CM" CM CM"
vo oo ^o oo oo oo
CM CM CM CM CM CM
•» A •» M •* *
\O O vO O O O
vO OO vO OO OO 00
CM CM CM CM CM CM
•vf OO <• OO OO 00
CM CM CM CM CM CM
TJ TJ
TJ CU TJ CU
CU 4J CU 4-1
4-1 Cfl 4-> Cfl
Cfl rH CO rH
9) >* 9) >,
VJ ,d t-i ^3
4J 4-1 4-1 4-1
fi CU C CU
TJ TJ 3 S 3 8
TJ TJ ^ ^ in in
C C
-------
Table 26. In both experiments bile contained by far the highest levels
of l^C of any of the tissues examined. The highest biliary concentrations
of C observed in the short-term study were about 100 to 250 times the
initial water concentration of C, while in the long-term study biliary C
was maintained at 500 to 1,400 times the average water concentration of ^C
for the duration of the exposure, and the ratio was about 100 for the entire
elimination period.
TABLE 25. UPTAKE AND ELIMINATION OF 14C-1,2,4-TCB
BY RAINBOW TROUT: SHORT-TERM EXPOSURE3
l^C-TCB compound and metabolites,
Time, P8/g or ml of tissue
h Muscle
Exposure 2 0.60b
±0.03
4 0.78
±0.05
8 0.91
±0.07
Elimination 10 0.61
±0.09
32 0.13
±0.02
56 0.04
±0.02
Liver
1.40
±0.45
1.13
±0.09
1.83
±0.21
1.36
±0.16
0.35
±0.05
0.07
±0.03
Blood
0.23
±0.06
0.37
±0.09
0.59
±0.08
0.08
±0.06
0.00
0.00
Bile
0.7
±0.2(4)
2.2
±0.3(3)
1.9
±0.3(3)
4.3
±1.6
0.9
±0.3
0.6
±0.1(4)
aStatic exposure, initial concentration of TCB 0.018 mg/liter.
Average ± S.E. of values from five fish which were sacrificed at each time.
In some cases the bile could not be sampled; the number of samples of bile
is indicated in parentheses.
Levels of C in muscle, liver, and blood dropped rapidly during the
elimination period in both studies, but in the long term study the C
remaining in the muscle and liver disappeared more slowly. The half-lives
of elimination are presented in Table 27. Because of the abrupt change in
rate of loss of tissue C in the long term study after 1 day of
elimination, 1 day of elimination was selected as the basis of comparison of
elimination rates in the two studies. The rates of elimination of C were
similar for muscle and liver during the first 24 h of elimination. Although
this same rate of elimination was maintained for the second 24 h period
after short term exposure, the rate of elimination of C after long term
88
-------
TABLE 26. UPTAKE AND ELIMINATION OF 14C-1,2,4-TCB
BY RAINBOW TROUT: LONG-TERM EXPOSURE3
Time,
day
Exposure 1
2
4
7
11
18
35
Elimination 36
37
39
42
57
71
Muscle
0.82b
±0.07
1.27
±0.15
1.44
±0.21
1.51
±0.17
2.80
±0.32
1.60
±0.15
1.54
±0.20
0.18
±0.02
0.16
±0.02
0.13
±0.01
0.13
±0.01
0.11
±0.02
0.05
±0.01
14C-TCB and
pg/g or ml
Liver
3.12
±0.11
3.76
±0.19
4.19
±0.16
4.21
±0.36
8.47
±0.36
4.92
±0.26
7.01
±0.52
0.58
±0.09
0.53
±0.07
0.40
±0.08
0.36
±0.07
0.28
±0.07
0.25
±0.03
metabolites
of tissue
Blood
0.37
±0.09
0.56
±0.14
0.53
±0.11
0.86
±0.26
1.78
±0.35
1.51
±0.23
0.34
±0.14
0.00
0.00
0.00
0.00
0.00
0.00
>
Bile
10.9
±2.5
14.4
±1.1
13.0
±2.1
16.4
±2.8(4)
9.1
±5.8(3)
24.2
±4.1
5.0
±4.5(3)
1.5
±0.8(3)
2.4(1)
1.6
±0.4
1.0
±0.4
1.6
±0.3
Exposure performed using a continuous flow delivery system; average water
concentration of TCB is 0.018 mg/liter.
"Average ± S.E. of values from five fish which were sacrificed at each time,
except day 71 when seven fish were sacrificed. In some instances the bile
volumes were inadequate for sampling; the number of samples of bile is
indicated in parentheses.
89
-------
exposure dropped to < 1% of the initial rate.
Blood C dropped to undetectable levels in both studies by 24 h of
elimination. The data after 2 h of elimination in the short term study
indicated a half-life of < 1 h for blood 14C.
Information on metabolites of ^C-1,2,4-TCB in bile and tissue extracts
is presented in Section 4 (Effect of Inducers on Disposition of Organic
Chemicals in Rainbow Trout).
TABLE 27. ELIMINATION OF TRICHLOROBENZENE BY RAINBOW TROUT
Exposure
8 h
35 days
Elimination
0-24 h
0-48 h
0-24 h
1-36 days
Half-life of
Muscle
9.0 h
10.9 h
7.7 h
36.1 days
elimination
Liver
10.5 h
10.5 h
6.7 h
32.2 days
Di-2-ethylhexylphthalate (DEPH)
Phthalate esters are a class of chemicals widely used and commonly
present in water and aquatic species. These chemicals can be made more
polar by hydroysis, but at the same time they may be more extensively
metabolized. The di-2-ethylhexyl ester is the most common and was selected
for study in vivo and in vitro.
Metabolism and Disposition In Vivo —
Three groups of rainbow trout were exposed to 0.5 ppm of ^C-DEHP as
described under Methods (Melancon and Lech 1976). The first group of 14
70-100 g fish yielded 191 yg of DEHP equivalents in the pooled bile.
Subsequent groups of 34 70-100 g fish and 48 180-240 g fish yielded 769 and
1634 yg of DEHP equivalents in the pooled bile from each exposure,
respectively. The tissue distribution of C expressed as DEHP equivalents
for the second exposure is given in Table 28. One-half of the total DEHP
and metabolites in the fish was found in the bile, and the concentration was
over 200 times that of DEHP originally present in the water.
Metabolites were isolated from pooled bile as shown in Figure 36. Bile
from each exposure was pooled and desalted with an XAD-2 column. Most of
the data presented here pertains to bile from the third exposure. The XAD-2
eluate contained 90.2% of the ^C applied to the column. An aliquot of the
90
-------
a is r-
2. Q
to
5
re
CU*-*i
—Id
«^ _
i-H
X
Q.
1°
So
-JX
•*•«> "
§
3.
£
>>
re
l*>7*l
00
»j Wl -O
o in X
Sit
CM
O »
X
lil-feC)
r*- a;*
5S i
0^5
•T^m1
I re o
°Ed
5*-s
g
3 tS
^ |?
u? S
^w .^
v -a
JC —
UJ
o
1
1-
*j "re "SJ
O O Ol
fits
III "* .
^ 5 :•=
10 X
sl
O »• -j; re
S "5 *
m
^
O
^J
t-
8
:§.
re
<
O
^j
1-
1
^
>,
re
5
<
•S
1
•S 3
*S
So
(/)V«J
QJ
l/>
-g
§
1,1
So
— 1
y)C^.
5
o
&
.oX
•ao
V
I
u
_re
o
i Sacchar
OO
e
-»• o -*% „
< IJ
£i/5
a.
— H
O
S
•s ^ N
if! *a
o o 5 .^ -^Lu —
' =5
U
i
Si
a.
"*" EJ o
^3
'o
<
Q)
(A
(TJ
*H |
CO o
1_
3
u
3
O
1
*H ^
CD
— »-
_ _
5
S
~
to
cJ
^J
1-
s
4-1
S
8.
0)
£
1
(J
re
ii
X
UJ
a.
LLj
-------
XAD-2 eluate was chromatographed with chloroform:methanol:acetic acid
(143:7:2)(solvent 2). As shown in the uppermost graph of Figure 37, this
material was much more polar than the DEHP starting material.
TABLE 28. TISSUE LEVELS OF DEHP (AND/OR METABOLITES) FOLLOWING
24 H EXPOSURE TO AQUEOUS DEHP AT AN INITIAL LEVEL OF 0.5
Tissue
Tissue weight, g
Total DEHP, ug
DEHP, yg/g
Bile
Blood
Liver
Gills
Fins
Viscera
Kidney
Skin
Head
Carcass
Whole fish
0.2
0.4
0.7
2.4
1.3
4.9
0.4
6.2
6.4
49.5
72.3
22.6
0.2
2.1
5.4
1.7
2.6
0.2
1.2
2.5
5.5
44.0
111.4
0.5
3.0
2.3
1.3
0.5
0.5
0.2
0.4
0.1
0.6
aValues for bile, blood, and liver are based on the pooled samples from 34
fish. Values for all other tissues are based on the pooled samples from
two fish.
Fractionation was performed as described in Figure 36 and resulted in
radioactive fractions A, B, C, and D which contained 1.5, 62.5, 18, and 18%,
respectively, of the XAD-2 eluate radioactivity. Suitably-sized aliquots of
Fractions A through D were chromatographed with solvent system 2. As shown
in Figure 35, Fraction A contained mostly unchanged DEHP (metabolite AI) and
a small amount of mono-2-ethylhexyl phthalate (I1EHP) (metabolite All), both
at such low levels that they were not observed in the earlier TLC of the
XAD-2 eluate. Fractions B, C, and D consisted of more polar materials.
It was anticipated that DEHP metabolites might be present in bile, and
the solubility and TLC mobility of the bile fractions suggested the presence
of conjugates. To check for the presence of glucuronides, aliquots of
Fractions B, C, and D were incubated with g-glucuronidase with and without
saccharo-l,4-lactone. As the data in Table 29 show, the high percentage of
the radioactivity in each fraction that remained near the origin was reduced
by 25-90% by 3-glucuronidase hydrolysis. In all fractions this hydrolysis
was reduced substantially by the presence of saccharo-l,4-lactone. Each of
the fractions was examined in more detail.
92
-------
SOLVENT 2
PA
800-
600-
400-
200-
0-
200-
0-
800-
600-
^ 400-
r>
Q 200-
0-
600-
400-
200-
0-
200-
n.,
•— ^- t^i_i H
XAD-2
ELUATE
^^•^^
FRACTION A
, [~|
I 1
FRACTION B
—
FRACTION C
^^^_^^^«^
-, FRACTION D
0 2 4 6 8 10 121416
CM FROM ORIGIN
Figure 37. Thin-layer chromatography of fractionated rainbow trout bile.
Solvent 2 (chloroformrmethanol:acetic acid of 143:7:2). The
mobilities of phthalic acid (PA), MEHP, and DEHP are indicated
by the dark spots.
93
-------
TABLE 29. 3-GLUCURONIDASE HYDROLYSIS OF MAJOR BILE METABOLITE FRACTIONS3
Total dpm remaining at origin, %
+ 3-glucuronidase
Fraction Control + 3-glucuronidase + saccharo-l,4-lactone
B
C
D
97.4
92.5
96.3
8.2
60.6
72.2
62.6
87.6
95.0
aTotal incubation volume was 0.5 ml containing 100 units of 3-
glucuronidaseand 0.001 M saccharo-l,4-lactone where indicated. Solvent
system 2 was used for TLC.
Preparative TLC of Fraction B with chloroform:methanol:acetic acid
(5:1:1) (solvent 4) resulted in three radioactive peaks. The major peak
(metabolite BII) had an R^ value of 0.61, and the minor peaks (metabolites
BI and BUI) had Rf values of 0.44 and 0.85, respectively. Samples of each
of the three metabolites were incubated with g-glucuronidase. These three
metabolites were chromatographed with solvent 4 before and after incubation
(Figure 38). Metabolites BI and BII were both changed to less polar
compounds by g-glucuronidase action, but metabolite Bill was unaffected. In
this solvent system metabolite BUI and hydrolyzed metabolites BI and BII
all co-chromatographed with MEHP. Because metabolites BI and BII
chromatographed differently from each other as glucuronides, we expected
that two different compounds would be released by 3-glucuronidase
hydrolysis. This was confirmed by chromatography with solvent 2. As shown
in Figure 39, hydrolyzed metabolite BII co-chromatographed with MEHP,
whereas both metabolites Bill and hydrolyzed BI were more polar. Hydrolyzed
metabolite BII, after reaction with diazomethane, co-chromatographed with
MeEHP, as shown in Figure 40. Metabolite BII accounted for about 56% of
bile radioactivity and appeared to be MEHP glucuronide. Metabolites Bill
and hydrolyzed BI were pooled because of their similar mobilities in TLC and
are referred to later.
Fraction C was characterized by use of the same techniques described
for the study of fraction B. This fraction was composed mainly of MEHP
glucuronide (metabolite CI).
Fraction D was incubated with 3-glucuronidase, followed by
acidification and ether extraction. Although this fraction—after
acidification—had been extracted exhaustively with ether during the
original fractionation, this procedure now yielded a compound (hydrolyzed
Metabolite DI) which was similar to Metabolites Bill and hydrolyzed BI.
Figure 41 shows that the Rf value of hydrolyzed Metabolite DI is different
from that of non-hydrolyzed Fraction D and similar to that of MEHP in
solvent 4, whereas its mobility in solvent 2 is definitely different from
94
-------
100-
o.
Q
400-
200-
400-
200-
BI
SOLVENT 4
PAMEHP
BIT
BET
0 2 4 6 8 101214 16
CM FROM ORIGIN
Figure 38. Effect of $-glucuronidase hydrolysis on the TLC mobility of
Metabolites BI, BII and Bill. Mobilities of Metabolites BI and
BII before and after 3-glucuronidase hydrolysis are represented
by open and closed bars, respectively. The striped bar for
Metabolite Bill indicates that the Rf value of this metabolite
was unchanged by incubation with 3-glucuronidase. Solvent 4 is
chloroform:methanol:acetic acid (5:1:1). The mobilities of PA,
MEHP, and DEHP are indicated by dark spots.
95
-------
PA MEHP
100-
600-
400-
200H
o
200 J
SOLVENT 2
DEHP
• BI
After
J-Glucuronidase
BE
After
3-Glucuronidase
BUI
0 2 4 6 8 10 12 14 16
CM FROM ORIGIN
Figure 39. Thin-layer chromatography of Metabolites BI, BII and Bill after
incubation with 3-glucuronidase. Solvent 2 is chloroform:
methanol:acetic acid (143:7:2). The mobilities of PA, MEHP, and
DEHP are indicated by dark spots.
96
-------
800-
Q_
Q
400-
MEHP
MeEHP DEHP
SOLVENT 1
PA
DMP
METHYLATED BH
AFTER
3-GLUCURONIDASE
0 2 4 6 8 10 12 14
CM FROM ORIGIN
Figure 40. Thin-layer chromatography of Metabolite BII, methylated following
B-glucuronidase hydrolysis. Solvent 1 (benzene:ethyl acetate
(19:1). The mobilities of PA, MEHP, DMP, MeEHP, and DEHP are
indicated by dark spots.
97
-------
200-
Q.
Q
400-
200-
0
400-
SOLVENT 4
MEHP
PA DEHP
Fraction D
DI
After
J-Glucuronldase
SOLVENT 2
PA MEHP DEHP
200-
n .
"1
DI
After
J-Glucuronidase
0 2 4 6 8 10 12 1416
CM FROM ORIGIN
Figure 41. Thin-layer chromatography of Fraction D (top graph), and of the
acidic ether extract of this fraction after 3-glucuronidase
hydrolysis (hydrolyzed Metabolites DI), lower graphs, in two
solvents. Solvent 4 is chloroform:methanol:acetic acid (5:1:1)
and was utilized to obtain upper graphs. Solvent 2 is chloro-
form:methanol:acetic acid (143:7:2) and was utilized to obtain
the lower graph. The mobilities of PA, MEHP and DEHP are
indicated by dark spots.
98
-------
that of MEHP and similar to that of metabolite Bill.
(Dili) also was found.
A small amount of PA
Because of their similar mobilities in TLC, metabolite Bill and
hydrolyzed metabolites Bl and DI were combined. Although reaction with
diazomethane decreased the polarity of this material, it was still at least
as polar as MEHP.
The radioactive material that remained in the aqueous phase after 3-
glucuronidase treatment of Fraction D and extraction contained 9% of the
total bile radioactivity. This material (metabolite DII) has been
characterized only by its relatively high polarity in TLC, resistance to g-
glucuronidase hydrolysis, and lack of extraction from aqueous solution by
the organic solvents used in this study.
A summary of the approximate amounts of DEHP and metabolites found in
the various bile fractions is presented in Table 30. Unchanged DEHP
accounted for only 1% of the bile radioactivity. About 83.5% of the
radioactivity was present as glucuronide conjugates—namely, MEHP
glucuronide, (72%); PA glucuronide, (2%); and polar metabolite glucuronide,
(9.5%). In addition, about 9% of bile radioactivity was present as very
polar material.
TABLE 30. DISTRIBUTION OF 14C IN FRACTIONATED TROUT BILEa
Compound
DEHP
MEHP
MEHP-glucuronide
PA -glucuronide
Polar metabolite
Polar metabolite-glucuronide
Polar-nonextractable material
Designation
AI
All
BII,CI
Dili
Bill
BI.DI
DII
Fraction, %
A B C
1
0.5
56 16
3
2.5
D
2
7
9
aFractions as described in text.
activity in XAD-2 eluate.
percentages are based on radio-
In a similar experiment, fractions of radioactivity from the XAD-2
eluates of bile from the first two exposure groups extractable into benzene
or acidic ether before and after $-glucuronidase hydrolysis were pooled.
Preparative TLC with several solvent systems yielded six radioactive
peaks. One of these co-chromatographed with DEHP, several co-
chromatographed with MEHP, and another was more polar than MEHP and was
99
-------
similar to the combined polar metabolites Bill and hydrolyzed BI and DI
described earlier. These metabolites were subjected to GC/MS and the
results are shown in Table 31. The GC peaks were located with the
radioactive monitor to insure that actual metabolites were being
identified. In agreement with the TLC data, the GC/MS results obtained with
sample 1 were consistent with the presence of unchanged DEHP. The results
with methylated samples 2, 3, 4, and 5 were consistent with the presence of
MeEHP, indicating MEHP as the major metabolite. Sample 6, after
methylation, chromatographed as more polar than any of the three standard
diesters and lacked a peak at m/e 112, corresponding to the 2-ethylhexyl
moiety, suggesting a metabolite with a modified sidechain.
TABLE 31. THE GC/MS ANALYSIS OF PHTHALATE METABOLISM
GC-RAM elution temperature, "C
MS (m/e+)
5% DECS
3% OV-7
Base
peak
Secondary
peak
Standards
DEHP
MeEHP
DMP
Samples
1
2a
3a
4a
5a
6a
225
203
174
225
203
203
203
203
240
231
189
133
189
189
190
240
149
163
163
149
163
163
163
167
149
167
149
149
149
Methylated.
These studies show that rainbow trout readily can convert DEHP to
MEHP. Table 32 compares the results of this study of DEHP and metabolites
in trout bile and of the study of whole catfish extracts following 24-h
exposure reported by Stalling et al. (1973). The 83.5% of biliary
phthalates found as conjugates (see Table 30) is > 14% of conjugated
phthalates present in whole fish extracts reported by Stalling et al.
(1973).
100
-------
TABLE 32. DEHP AND METABOLITES IN RAINBOW TROUT AND CATFISH
Rainbow trout,3 Catfish,b
Compound % in bile % in whole fish
DEHP
MEHP
MEHP-glucuronide
PA
PA-glucuronide
Other
1
0.5
72
0
2
21.5
14
66
13.7
4
0.3
2
aRainbow trout data summarized from Table 30.
bCatfish data from Stalling et al. (1973).
Although it was expected that the level of conjugates in bile would be
higher than in whole fish, such a large difference was not expected.
Because bile radioactivity represents about 50% of total fish radioactivity
in this study, the finding of 83.5% biliary conjugates is equivalent to >
40% conjugates on a whole-fish basis, even if no conjugates are present in
the remainder of the fish. Expressed in terms of quantities of each
phthalate metabolite, the results agree more closely. Total MEHP accounted
for about 72.5% in rainbow trout bile as compared to 80% in whole catfish.
The large difference in total conjugates between the two species suggests
either that the two species differed in rate of conjugation or that
enterohepatic circulation and redistribution occurred in the catfish
studies.
Total PA was low in both species. Significant differences were
observed, however, in the amounts of unchanged DEHP and of very polar
metabolites. It was expected that bile would contain mostly DEHP
metabolites, whereas the other tissues would be more likely to contain
unmodified DEHP. However, the 21.5% of very polar metabolites found in trout
bile is equivalent to > 10% on a while-fish basis, i.e., five times the
value reported for catfish. The difference in amounts of these very polar
metabolites might be caused by species differences but also could occur
because the trout were exposed to 0.5 ppm DEHP whereas the catfish were
exposed to only 0.001 ppm. The effect of exposure level on metabolite
patterns is under investigation.
Possible pathways for the formation of DEHP metabolites found in
rainbow trout bile are illustrated in Figure 42. From the large amount of
MEHP glucuronide present in rainbow trout bile, the sequence DEHP^IEHP-MEHP
glucuronide was obviously the major metabolic pathway. The PA glucuronide
could have arisen by either of the indicated pathways. The unidentified
polar glucuronide could have been formed in a variety of ways. The mass
spectral data suggest that the ring structure was intact whereas the 2-
101
-------
X
o — t
\
o
1
o
1
o
1
o
1
J — O
1
o
1
o
1
o
1
o
1
o
1
{_>
1
o — <_> — o
1
O _
1
o
1
X
\
O=
o
1
0
1
o
o
1
o
1
o
1
o
1
o — o — o
o
1
0
1
o =o
CO -H
Csl
-*
(U
!-l
102
-------
ethylhexyl moeity was modified. Several of the studies of DEHP metabolism
in rats have indicated that DEHP is hydrolyzed to MEHP before the 2-
ethylhexyl moeity is modified. Because glucuronide formation generally is
considered to facilitate excretion, MEHP glucuronide is probably not further
metabolized by oxidation. These two considerations suggest that the pathway
for the formation of this metabolite is DEHP-MEHP-oxidized MEHP-oxidized
MEHP glucuronide.
Metabolism of DEHP in Rainbow Trout In Vitro—
DEHP metabolism was initially studied in vitro by utilizing the 2000 g
supernatant fraction of trout liver homogenates prepared in 0.154M KC1
(Melancon and Lech 1977). Representative radioscans of thin-layer
chromatograms of extracts of incubations of carboxyl-labeled ^C-DEHP with
the 2000 9 supernatant fraction are shown in Figure 43. The lower tracing
shows that when NADPH was not added to the incubation mixture, MEHP was the
predominant metabolite formed. Addition of NADPH, shown in the upper
tracing, resulted in the appearance of two additional radioactive peaks.
The peak adjacent to that of MEHP was designated polar metabolite 1 and the
peak near the origin, polar metabolite 2, inasmuch as they have not been
identified.
A summary of the amounts of MEHP and polar metabolite 1 formed in
several such experiments is presented in Table 33. Without added NADPH,
metabolism consisted almost entirely of hydrolysis of DEHP to MEHP. When
NADPH was added the production of polar metabolite 1 increased
substantially. The increase in polar metabolites, a decrease in MEHP
accumulation, and an increase in total DEHP metabolism resulting from the
addition of NADPH are shown in Table 33, and are also apparent in Table 34
and Figure 44 and Figure 45. Piperonyl butoxide blocked hydrolysis and
oxidation of DEHP. Hydrolysis or oxidation of DEHP was not observed in
incubations containing heat-inactivated liver fractions.
TABLE 33. METABOLISM OF DEHP BY TROUT LIVER HOMOGENATESa
Additions
MEHP formed
nmoles/h/g of liver
Polar metabolite I formed
nmoles/h/g of liver
0
+ NADPH
+ PBO
+ NADPH, PBO
2.84 ± 0.68
1.62 ± 0.41
0.14 ± 0.00
0.00
0.27 ± 0.14
2.43 ± 0.68
0.00 ± 0.00
0.00
alncubations were performed as described in text.
0.034 moles 14C-DEHP in a total volume of 1 ml.
mean + SEM for three separate homogenates.
Each incubation contained
Values presented are the
103
-------
0
DEHP
+NADPH
PM2
PM1 MEHP
CONTROL
0
Figure 43. Metabolite patterns following incubation of ^C-DEHP with trout
liver homogenate. The 2000 g supernatant fraction of trout liver
homogenate was incubated as described in the text with 0.010 umol
of llfC-DEHP in a total volume of 2 ml for 1 h with or without
added NADPH. The ether extracts from the acidified incubation
media were subjected to TLC with chloroform:methanol:acetic acid
(143:7:2). DEHP and MEHP were located with chromatographed
standards. PM1 and PM2 represent Polar Metabolites 1 and 2.
104
-------
TABLE 34. METABOLISM OF DEHP BY SUBCELLULAR FRACTIONS OF
TROUT LIVER HOMOGENATES AND BY TROUT BLOOD SERUM3
Nanomoles of metabolite formed/h/g
of liver by addition of
Experiment Fraction NADPH PBO
1 2,000 g
supernatant
1 Mitochondria
1 Microsomes
1 100,000 g
supernatant
2 2,000 g
supernatant
2 Mitochondria
2 Microsomes
2 100,0005-
supernatant
Nanomoles
Serum
0
X
0
X
X
0
X
X
0
0
0
0
0
0
0
0
0
of
0
0
0
0
0
0
X
0
0
X
0
0
X
0
X
0
X
0
X
metabolite
0 0
X 0
Polar
MEHP metabolite 1
3.91
3.11
1.99
2.19
0.13
4.51
2.23
0.13
4.13
3.84
0.26
2.52
0.20
2.50
0.26
3.15
0.33
0.53
3.38
0.07
0.73
0.13
0.20
2.93
0.26
0.07
0.20
0.00
0.26
0.00
0.00
0.00
0.07
0.07
formed/h/0.8 ml of
.61±0.11
.0310.01
0.01±0.00
0.00
Polar
metabolite 2
0.20
1.66
0.07
0.33
0.07
0.13
1.41
0.07
0.07
0.07
0.00
0.07
0.00
0.07
0.07
0.07
0.07
serum
0.04±0.03
0.00
1 Incubations of 1-h were performed as described under Methodology. Each
incubation contained 0.010 ymoles C-DEHP in a total volume of 2 ml.
Serum samples from each of three fish were incubated separately. Values
for serum are the mean ± SEM.
105
-------
O)
Mitochondia
CONTROL + NADPH
Microsomes
CONTROL +NADPH
-------
S 6
o
I
Ti
6 4
2
Q>
O
I °
Mitochondria
CONTROL +NADPH
Microsomes
CONTROL +NADPH
THTT
1.252.5 5 1.252.5 5 1.252.5 5 2.5 5
DEHP concentration (nmoles/ml)
Figure 45.
Influence of DEHP concentration on metabolism of JltC-DEHP by
trout liver mitochondrial and microsomal fractions. Incubations
as described in the text, were done in a total volume of 2 ml
for 1 h. Mitochondria equivalent to 0.254 g of liver of micro-
somes equivalent to 0.361 g of liver were used in each incubation.
Open bars represent MEHP, striped bars Polar Metabolite 1 and
solid bars Polar Metabolite 2. Each column represents an
individual incubation.
107
-------
In subsequent experiments the 2000 g supernatant fraction was further
fractionated as described in the section on methodology to determine which
organelles were reponsible for DEHP hydrolysis and oxidation. Because
blood—including fish blood—is known to contain esterase activity
(Augustinsson 1959), the ability of trout serum to catalyze DEHP hydrolysis
was also investigated. Ring-labeled C-DEHP was used in these and all
following experiments. The results of two experiments comparing the
metabolism of DEHP by the 2000 g supernatant fraction and by the
"mitochondrial", "microsomal", and 100,000^ supernatant fractions are
presented in Table 34. All the liver homogenate fractions showed
significant hydrolysis of DEHP to MEHP, which was inhibited by PBO.
Incubation of the mitochondrial or microsomal fraction with DEHP and NADPH
gave results similar to those obtained with the 2000 g supernatant fraction.
Although not shown in Table 34, added NADPH had little effect on DEHP
metabolism by the 100,000 g- supernatant fraction. Serum contained DEHP-
hydrolytic activity that also was blocked by PBO.
Figure 44 and Figure 45 show the time-course and substrate dependence
of DEHP metabolism by the "mitochondrial" and "microsomal" fractions with
and without added NADPH. In addition to demonstrating the increased
production of metabolites with increasing incubation time and DEHP
concentration, the data show clearly the effects of addition of NADPH, i.e.,
a greatly increased production of polar metabolites 1 and 2 with reduced
accumulation of MEHP.
The ability of PBO to block formation of the oxidized metabolites of
DEHP was not surprising, but its inhibition of the hydrolysis of DEHP to
MEHP—both in liver fractions and in serum—was unexpected. To investigate
this effect further, the metabolism (hydrolysis) of a different ester, 2,4-
DBE, by these liver fractions and blood serum was measured. Studies have
shown that 2,4-DBE is hydrolyzed readily by fish in vivo (Rodgers and
Stalling 1972, Statham and Lech 1976). The data in Table 35 show that trout
liver subcellular fractions readily hydrolyze 2,4-DBE and that PBO
substantially inhibits the hydrolysis. Also, PBO inhibited the serum-
catalyzed hydrolysis of 2,4-DBE.
In a number of experiments in which the metabolism of DEHP by the 2000
g supernatant fraction and by subtractions prepared from it was measured,
the hydrolysis of DEHP by the 2000 g supernatant fraction was not great
enough to account for the sum of DEHP esterase activities present in the
mitochondrial, microsomal, and 100,000 £7 supernatant fractions.
In order to investigate this observation, a 2000 g supernatant fraction
of liver homogenate was centrifuged at 100,000^, and this pellet was
resuspended in a volume of 0.154 M KC1 equal to 25% of the volume of the
supernatant fluid removed. Varying amounts of this suspension and of the
100,000 g supernatant fraction were incubated in the usual manner, i.e.,
separately and in combination with each other. The results presented in
Table 36 show no linear increase in amount of DEHP hydrolyzed with
increasing amounts of the particulate suspension and show that the
hydrolytic activity of the combined fractions does not equal the sum of the
hydrolytic activities of the fractions individually.
108
-------
TABLE 35. METABOLISM OF 2,4-DBE BY SUBCELLUAR FRACTIONS
OF TROUT LIVER HOMOGENATES AND BY TROUT BLOOD SERUMa
Nanomoles of 2,4-DBE hydrolyzed/h/g of liver by addition of
2,000£ 100,0000
Experiment NADPH PBO supernatant Mitrochondria Mlcrosomes supernatant
1
2
0
X
X
0
0
0
0
X
0
X
101.4
100.0
28.4
97.3
33.1
92.6
79.1
29.7
103.4
19.6
107.4
100.0
25.0
86.5
28.4
83.8
108.1
30.4
Serum
Nanomoles of 2,4-DBE hydrolyzed/h/0.08 ml of serum
0
0
0
X
16.0 ±
2.2 ±
0.4
0.3
The 1-h incubations were performed as described under Methodology. Each
incubation contained 0.02 umoles of 1^C-2,4-DBE in a total volume of 2
ml. Serum samples from each of three fish were incubated separately.
Values for serum are the mean ± SEM.
Because of the occurrence of DEHP-metabolizing activity in the various
liver homogenate fractions and because of the close similarity between
mitochondrial and microsomal metabolism of DEHP shown in Figure 42 and
Figure 43, these fractions needed to be characterized further. For this
reason, the activity of marker enzymes and DEHP metabolizing enzymes and
protein concentration were determined for the 2000 g , 10,0000, 100,0000
pellets and 100,000 0 supernatant fraction of trout liver homogenates. The
enzyme activities measured were succinic dehydrogenase, glucose 6-
phosphatase, DEHP esterase (MEHP formation without added NADPH), and DEHP
oxidase (formation of metabolites more polar than MEHP with added NADPH).
The relative specific activity for each enzyme activity measured in each
fraction versus the percentage of total protein present in each fraction as
described by deDuve et al. (1955) is presented in Figure 46.
The data concerning the distribution of the mitochondrial marker
(succinic dehydrogenase) and the microsomal marker (glucose 6-phosphatase)
indicate that the standard rat liver fractionation technique does not
produce a satisfactory separation of organelles when trout liver is used.
Based on succinic dehydrogenase activity, most mitochondria were present in
the 2000 0 pellet, with a smaller fraction in the so-called "mitochondrial
fraction." Some microsomes (glucose 6-phosphatase) were distributed in the
2000 0 pellet, but high relative specific activity was observed in the
"mitochondrial" as well as in the "microsomal" fractions. In addition, DEHP
109
-------
o
«J
0)
JS
0)
3-
2-
Succinic Dehydrogenase
A B C D
20 40 60 80 100
Glucose-6- Phosphatase
20 40 60 80 100
DEHP Esterase
20 40 60 80 100
DEHP Oxidase
0 20 40 60 80 100
Percentage of total protein
Figure 46. Distribution of marker enzymes and DEHP-metabolizing enzymes
in trout liver homogenate fractions. DEHP esterase and DEHP
oxidase were each measured by 1 h incubations of 0.010 ymol of
^C-DEHP in a total volume of 2 ml. A. 2000 g pellet:
B. 10,000 g pellet: C. 100,000 g pellet: D. 100,000 g
supernatant fraction. Relative specific activity - percentage
of total activity percentage of total protein.
110
-------
TABLE 36. HYDROLYSIS OF DEHP BY RECOMBINED
TROUT LIVER HOMOGENATE FRACTIONS3
Hydrolysis rate (nmole/h) for liver
equivalent of mitochondria and
Liver equivalent of 100,000 g microsomal fractions of,
supernatant fraction, mg 0 33 mg 66 mg 132 mg
0
68
136
0.03
0.45
1.11
0.39
0.70
(0.89)
0.75
(1.50)
0.46
0.94
(0.91)
0.94
(1.57)
0.59
0.74
(1.04)
0.92
(1.70)
aIncubation conditions were as described in the text except that a highly
concentrated resuspension of the mitochondrial plus microsomal pellet was
used so that the volumes of this suspension plus 100,000 9 supernatant
fraction could be kept to 0.8 ml. Each reaction vessel contained 0.01
umole of 14C-DEHP in a volume of 2 ml. Incubation was for 1 h at 22°C.
All incubations containing 136 mg (liver equivalent) of 100,000 g
supernatant fraction were performed in duplicate, wherease the other data
are individual values. Expected values are given in parentheses.
oxidase activity is high in the fractions that contain most of the glucose
6-phosphatase and low in the 2000 g pellet, which contains most of the
mitochondria. The DEHP esterase activity was high in the fractions which
were high in glucose 6-phosphatase and DEHP oxidase activities, and
substantial esterase activity occurred in the 100,000 g supernatant
fraction, which contained little if any mitochondrial or microsomal markers.
The results show that rainbow trout liver homogenates have the ability
to metabolize DEHP. Without added NADPH, metabolism consisted mainly of
hydrolysis of DEHP to MEHP. Whereas with added NADPH metabolism shifted
from production of MEHP to more polar metabolites and more total DEHP was
metabolized.
The production of polar metabolites in the presence of added NADPH
could depend on oxidation of DEHP itself or on oxidation of MEHP. The
reduced level of MEHP after incubations with NADPH as compared to those
without NADPH suggests that the polar metabolites appear at the expense of
MEHP. In most incubations < 20% of the DEHP was metabolized; thus,
sufficient DEHP was present as substrate for the hydrolytic reaction. These
data suggest that oxidation of MEHP occurs subsequent to hydrolysis of
DEHP. Studies of the in vivo metabolism of DEHP in rats demonstrated that
111
-------
except for a small amount of phthalic acid, the metabolies were either I1EHP
or oxidized derivatives of MEHP (Albro et al. 1973, Daniel and Bratt
1974). Similar results were observed with other phthalate esters (Albro and
Moore 1974, Williams and Blanchfield 1975). The absence of oxidized diester
metabolites suggests that metabolism of these phthalate esters consists of
hydrolysis to a monoester followed by oxidation of the remaining side chain,
or that any oxidized diester produced is very rapidly hydrolyzed to the
oxidized monoester. The results of in vitro metabolism of 14C-DEHP by
channel catfish liver microsomes reported by Stalling et al. (1973) indicate
that added 14C-MEHP was not further metabolized. Identification of the
polar metabolites of DEHP formed by liver fractions from rainbow trout and
catfish may explain the apparent differences between these studies.
Both DEHP and 2,4-DBE were hydrolyzed by all of the liver homogenate
fractions and by serum; hydrolysis of both compounds was inhibited by PBO.
Other researchers have reported that PBO can inhibit the microsome-catalyzed
hydrolysis of the pesticides parathion and diazinon but that this hydrolysis
required NADPH and oxygen (Nakatsugawa and Dahm 1967, Nakatsugawa et al.
1969). In this study, NADPH was not required for DEHP hydrolysis, but
hydrolysis by the liver fractions and by serum was inhibited by PBO.
Although no apparent explanation exists for this inhibition, a similar
effect has been observed in vivo with dioctyl phthalate in Culex and
Estigmene aarea larvae (Sanborn et al. 1975).
When rates of hydrolysis of DEHP and 2,4-DBE by liver homogenates and
by serum were compared per gram of liver and per milliliter of serum, the
rate of hydrolysis by serum was twice that of liver. Host of the
experiments in the current study were performed at approximately 2 ppm
DEHP. In vivo exposure of trout to DEHP at 0.5 ppm in water for 24 h gave
3.0 ppm in liver and 0.5 ppm in blood (Melancon and Lech 1976). Thus, the
DEHP levels in vivo and in these in vitro studies of DEHP metabolism were
similar.
When increasing amounts of the particulate fractions were added to the
100,000 g supernatant fraction, a linear increase in DEHP hydrolysis was not
observed. At the higher level of 100,000 g supernatant fraction used,
addition of the particulate fraction actually caused an inhibition of
hydrolysis. Because of the lipophilic nature of DEHP, DEHP may be non-
specifically bound by these organelles. In fact, the in vivo accumulation
of DEHP by beef heart mitochondria has been reported (Nazir et al. 1971).
Although DEHP esterase activity occurred in all the liver subcellular
fractions, the distribution of the microsomal marker—glucose 6-phosphatase-
-obviously demonstrated that DEHP esterase was distributed with microsomes
and was also present in the 100,000 g supernatant fraction. Although DEHP
oxidase was also present in several fractions, this activity was highest in
fractions that were highest in glucose 6-phsophatase activity. The studies
of Carter et al. (1974) with rat liver subcellular fractions demonstrated
the hydrolysis of DEHP in "mitochondrial" and "microsomal" fractions and a
trace of activity in the cytosol. Because the mitochondrial fraction in
this latter report was sedimented by an 18,000 g centrifugation for 20 min,
microsomal contamination probably occurred in this fraction, and the DEHP
112
-------
metabolizing activity attributed to mitochondria may well reside in
microsomes. The accumulation of DEHP in vivo in beef heart mitochondria
(Nazir et al. 1971) also would suggest that mitochondria do not metabolize
DEHP.
The distribution of DEHP esterase activity in rainbow trout liver
subcellular fractions is interesting because it differs from that found in
rat liver preparations. Several reports (Underhay et al. 1956, Shibko and
Tappel 1964, Schwark and Ecobichon 1968, Ljungquist and Augstinsson 1971)
give the total esterase activity in cytosol as from < 1 to almost 20%. The
higher values generally are reported when microsomal contamination of the
cytosol is present (Underhay et al. 1956, Schwark and Ecobichon 1968). In
the experiment shown in Figure 44, 34.6% of total DEHP esterase activity and
0.4% of total glucose 6-phosphatase activity were found in the 100,000 g
supernatant fraction, and in a subsequent experiment the values were 30.7%
and 1.6%, respectively. Thus, residual microsomal activity in the 100,000 g
supernatant fraction cannot account for the high esterase activity found in
this fraction from trout liver. Possible explanations of this activity are
1. that two different esterases can hydrolyze DEHP and 2. a single esterase
may be partially solubilized from microsomes during preparation of liver
homogenates.
Effect of PBO on Metabolism of Di-2-ethylhexylphthalate In Vivo and
In Vitro—
The inhibiting effect of PBO on the metabolism of DEHP and 2,4-DBE in
Vitro was not explained by the reported effects of PBO. Additional
experiments were performed to examine the in vitro effects in more detail
and to determine whether this PBO inhibition of DEHP metabolism by fish also
occurred in vivo (Melancon et al. 1978).
Radiochromatogram tracings of the ether extracts of incubations of l^C-
DEHP with the 2000 g supernatant of trout liver homogenates with or without
PBO are shown in Figure 47. The upper tracing shows the major metabolite
peaks—I1EHP and polar metabolite 1—which were present when the incubations
contained added NADPH. The actual percentages of total C on the plate were
MEHP (6.2%); polar metabolite 1 (13.3%); and polar metabolite 2 (1.5%). The
lower tracing does not show any metabolite peaks from the incubation with
PBO present; the DEHP peak represented 99.5% of the total radioactivity
found on the plate.
A summary of the effects of 2 x 10~3 M PBO on DEHP and 2,4-DBE
metabolism in incubations with trout liver fractions and with serum is
presented in Table 37. The inhibition of hydrolysis of these compounds by
PBO in all fractions is readily apparent as is the inhibition of NADPH-
dependent DEHP oxidation by PBO in liver fractions.
The effect of 9 x 10~3 M PBO on DEHP metabolism by trout liver
homogenate and serum is shown in Table 38. Even though the concentration of
PBO was lowered by a factor of over 22, hydrolysis of DEHP by serum and
113
-------
0
DEHP
- Piperonyl Butoxide
PM2 PM1 MEHP
+ Piperonyl Butoxide
z
CD
QC
O
Figure 47. Effect of PBO on the metabolism of DEHP by trout liver homo-
genates. The 2000 g supernatant fraction of a rainbow trout
liver homogenate was incubated in phosphate buffer with 5 ]\M
llfC-DEHP and 2 mM NADPH. llfC extracted following the incubation
was examined by TLC on silica-gel coated plates using
MeOH:HOAc (143:7:2) as solvent system. Tracings were obtained
using a radiochromatogram scanner.
114
-------
w
PQ
?
ft
CO
PQ
^
3 rS
c_>
o
w
Ǥ
Pi
f*
PQ
W
CQ
O
MH
ft
CO
cu
4-1
•iH
O
cfl
J
cu
N
0 60
43 CU
W O
?B
CM
o
cfl
4-1
CU
a
w
s
O S;
PQ B
ft CN
CS|
C
CU O
3 -r(
CO 4-1
CO O
•H Cfl
H M
M-l
CTirOOOO
OXOX
ooxx;
CU 4-1
4-1 Cfl
co a
a M
cu cu
60 P<
O 3
8 CO
O
cu o
> o
•H •>
i-J CM
CTi sf O O
v£> 00 O to
ooxx
CU 4-1
4-1 CU
CO rH
C rH
cu cu
60 a
o
o
42 O
O
M O
d) *
> o
•H O
O O
O O
-* CM
>J3 CM
T3
CU
N
O 60
M '
g I
P fl
CN
OOrHO OOCMO
O O O O OOOO
-H-H-H-H HH-H-HHH
i-H O -d" O ,-H O O O TJ
O O rH O OOrHO a
o
MH
43 42 43
OOOO OOvOO K60
• ••• . . . . pq-^
OOCMO OOOO P 42
-H-H4»-H -HHH-H-H MH
o o
CO OOOO OOOrH COS
CU C
OOvOO O O •* O 4J
•H
rH
O
XI 43 43 -Q 4-1
" " " I CU rH
rH O
rHOOO oddd 00
HH-H44-H -H-H-H-H -H
H CO
CO
e c
cfl O
0) -iH
a 4-1
a
CU CO
cfl m
3 CU
O >
43 i-t
CO rH
CO >*
CU 43
3
rH W
£§
W CNI
PQ
P MH
I O
•> CO
CM .H
O
O M
~ M
ft O
W ^
P 4J
^ CU
3. o
U1 CU
43 CO
4-1 C
•H O
S -H
4-1
4= CO
M
r-f Cfl
Cu
M CU
O M
MH d.
ou g
CM CO
CM CO
O
•
O
\s
a.
PQ
ft
• o
T3 M
CU 01
O
MH rH
M O
0) M
a, 4J
C
cu o
M O
a)
§
co
4-1 4-J 4-1 MH
co d
a) cu 4-j
•a w a C
CU CO -H CU
4-1 M M M
cfl CO 0) CU
43 (X CX MH
3 CU X MH
O CO O) -H
C TJ
•H CU O
0) S >~.
CU M 4J rH
01 4-J >-. C
IS rH CO
4-1 C! O
CO CO O vH
0) cfl MH
3 CU 43 -H
CO
CO
4J
co
-------
liver homogenate and oxidation of DEHP by liver homogenate were reduced by
about 90.
TABLE 38. EFFECT OF PIPERONYL BUTOX1DE ON METABOLISM OF
DEHP BY TROUT LIVER HOMOGENATE AND BLOOD SERUM
Additions
Metabolites of DEHP formed
nmole/h/g
Tissue
fraction
Liver homogenate
2,000 g
supernatant
Serum
NADPH
(2mM)
0
0
X
X
0
0
PBO
(90 yM)
0
X
0
X
0
X
Polar
MEHP
7.51 ± 0.82
1.05 ± 0.18b
1.14 ± 0.54
1.39 ± 0.37
15.96 ± 0.96
1.08 ± 0.12b
Polar
metabolite 1
0.22 ± 0.13
0.29 ± 0.18
11.29 ± 0.93
0.64 ± 0.33b
metabolite 2
0.14 ± 0.02
0.06 ± 0.01b
1.41 ± 0.46
0.08 ± 0.01b
aTissue were incubated at 22°C for 1 h with 5 ym DEHP. Data are presented
as mean ± SEM for four separate liver preparations and three serum samples.
bSignificantly different from control (zero PBO), p < 0.05.
Despite this inhibition by PBO of the in vitro metabolism of DEHP
(oxidation and hydrolysis) and 2,4-DBE (hydrolysis), we could not conclude
that PBO would necessarily have the same effects in vivo- Consequently, the
effect of exposure of trout to PBO upon in vitro metabolism of DEHP was
investigated. Liver homogenates were prepared from control fish and from
fish preexposed to 1 mg/liter PBO for 24 h. The data presented in Table 39
show a reduced production of the metabolites of DEHP by liver homogenates
from the fish preexposed to PBO. In the absence of added NADPH the
production of MEHP from DEHP was reduced, while in the presence of NADPH the
production of polar metabolites was reduced, suggesting that oxidation and
hydrolysis of DEHP were inhibited.
Another series of experiments was designed to determine whether
exposure of rainbow trout to PBO affected the disposition or metabolism of
DEHP in Vivo. Control fish were exposed to 0.07 mg/liter 14C-DEHP for 24 h,
and fish preexposed to 1 mg/liter PBO for 24 h were subsequently coexposed
to 0.07 mg/liter 14C-DEHP plus 1 mg/liter PBO for 24 h. The levels of
phthalates present in selected tissues expressed as microgram DEHP per gram
are presented in Table 40. Fish pretreated with PBO had muscle levels of
phthalate twice those of control fish and bile levels 50% those of control
fish. The concentration of phthalate also was elevated significantly in
blood in the PBO-treated fish. To determine whether the phthalates
accumulating in muscle were DEHP or metabolites, pooled muscle samples from
control and PBO-exposed fish from each experiment were extracted with
acetone and the extracted C was examined by TLC. The results presented in
Table 41 show that while almost 50% of the total phthalates present in
muscle of control fish was found to be MEHP, the value for MEHP in PBO-
exposed fish was < 25% of the total phthalate residue.
116
-------
TABLE 39. METABOLISM OF DEHP BY 2000 X G SUPERNATANT OF LIVER
HOMOGENATES FROM CONTROL TROUT AND TROUT PREEXPOSED TO PBOa
Metabolites of DEHP formed
nmole/h/g
Assay conditions
Treatment NADPH (2 mW
Control 0
+
Piperonyl 0
butoxide (1 mg/liter) +
9.
3.
3.
2.
MEHP
16
15
95
35
•t
±
±
±
1.58
0.63
0. 21b
0.31
Polar
metabolite 1
0.
7.
0.
2.
72
68
15
63
±
±
±
±
0.50
0.57
0.09
1.28b
Polar
metabolite 2
0.11
1.57
0.06
0.15
±
±
±
±
0.05
0.61
0.01
0.04
aTissues were incubated at 22°C for 1 h with 5 M DEHP. Each value is the
mean ± SEM for three liver homogenates.
bSignificantly different from control, p < 0.02.
TABLE 40. EFFECT OF PBO ON ACCUMUATION OF
14C-DEHP IN VARIOUS TISSUES OF RAINBOW TROUT IN VITROa
Treatment
C concentration,
Muscle Blood
pg/g DEHP
Bile
Liver
Control 0.021 ± 0.003 0.142 ± 0.017 51.4 ± 5.5 0.86 ± 0.08
PBO (1 mg/liter) 0.041b ± 0.006 0.234b ± 0.019 26.2 ± 2.8b 1.08 ± 0.15
aControl trout and trout which had been exposed to 1 mg/liter of PBO for
24 h were exposed respectively to 0.07 mg/liter 14C-DEHP and to 0.07
mg/liter 14C-DEHP plus 1 mg/liter of PBO for 24 h.
bSignificantly different from control, p < 0.01.
TABLE 41. EFFECT OF PBO ON ACCUMULATION OF
DEHP AND MEHP IN MUSCLE OF RAINBOW TROUT IN VIVOa
Experiment
1
2
Treatment
Control
PBO (1 mg/liter)
Control
PBO (1 mg/liter)
Total
As DEHP
46.6
76.5
45.8
70.2
14C, %
As MEHP
41.8
18.4
37.1
26.0
Concentration
, Mg/g
As DEHP As MEHP
0.014
0.042
0.006
0.020
0.012
0.010
0.005
0.007
aControl trout and trout which had been exposed to 1 mg/liter of PBO for
24 h were exposed respectively to 0.07 mg/liter ^C-DEHP and to 0.07
mg/liter 14C-DEHP plus 1 mg/liter of PBO for 24 h. Muscle 14C was
characterized by TLC.
117
-------
When these percentages were applied to the total phthalate concentrations in
muscle, MEHP levels were similar in the control and PBO-exposed fish while
the differences in DEHP account for the difference in total muscle
phthalates.
The ability of PBO to block DEHP oxidation by trout liver in vitro
corresponds to the known effects of PBO to inhibit microsomal oxidations in
mammals (Casida et al. 1966). The effect of PBO in vitro to block the
hydrolysis of DEHP and 2,4-DBE by trout liver subcellular fractions and
trout serum was not anticipated, based on reported actions of PBO. The
inhibitory effect of PBO on the microsomal-catalyzed hydrolysis of parathion
and related compounds has been reported (Nakatsugawa and Dahm 1967,
Kamienski and Murphy 1971), but the proposed pathway of parathion metabolism
requires oxidation dependent on NADPH and On prior to hydrolysis.
Preexposure of trout to PBO resulted in reduced oxidation and
hydrolysis of DEHP by the 2000 g supernatant of trout liver homogenates,
indicating that these effects of PBO might be occurring in vivo
Preexposure of trout ot PBO in Vivo modified the disposition and
matabolism of DEHP in V-ivo, and the major effects observed were decreased
biliary phthalates and increased muscle phthalates in PBO-preexposed trout
as compared to controls. Examination of the phthalates in muscle revealed
that the level of DEHP in this tissue from PBO-preexposed fish was three
times that of controls.
Thus, the in vivi and in Vitro effects of PBO on DEHP metabolism by
rainbow trout appear to be similar. As discussed previously, a decreased
oxidation of DEHP is consistent with the known effects of PBO to inhibit
microsomal oxidations. The reported inhibition of DEHP hydrolysis differs
from the indirect effect of PBO on hydrolysis of parathion in that the
hydrolysis of DEHP appears not to require NADPH and in that non-microsomal
hydrolysis of DEHP by the 100,000 g supernatant of liver homogenates and by
serum also was inhibited. Piperonyl butoxide has been shown to inhibit or
stimulate microsomal enzymes depending on the duration of treatment
(Kamienski and Murphy 1971). Because of the immediacy of esterase
inhibition seen in the in vitro experiments in this report, this effect is
obviously not related to long-term processes involving enzyme synthesis.
The two types of experiments reported herein involving in vivo exposures of
trout to PBO yielded data which were consistent with the in vitro results.
Liver homogenates prepared from PBO-exposed trout had lower DEHP esterase
activity based upon in vitro assays. In addition, the metabolic disposition
of DEHP in intact rainbow trout was altered by PBO in a manner which would
be consistent with the inhibition of DEHP hydrolysis in vivo*
The ability of PBO—which is not considered a direct inhibitor of
hydrolysis—to inhibit the hydrolysis of DEHP and 2,4-DBE raises the
question of the basis of this inhibition. The structures of DEHP, 2,4-DBE,
and PBO (Figure 48) indicate that the three compounds have a benzene ring
with at least one long chain substituent containing six or more carbon atoms
and oxygens in ester or ether linkage. Similarity also existed among the
phthalate moiety of DEHP, the methylenedioxyphenyl moiety of PBO, and the
118
-------
Di-2-ethylhexyl phthalate
Tropital
Piperonal
2/4-dichtoropfienoxy acetic acid -n-butyl ester
,CH
Piperonyl butoxide
Piperonyl alcohol
N-/ \_o_p_©_c2Hs)2
\—/
Paraoxon
Safrole
Met hytanadioKytaenzenc
Figure 48. Chemical structures of di-2-ethylhexylphthalate, 2,4,-dichloro-
phenoxyacetic acid-n-butyl ester, paraoxon and methylenedioxy-
phenyl compounds.
119
-------
dichlorophenyl moiety of 2,4-DBE. In order to assess whether the
methylenedioxyphenyl group or the side chain of EBO was responsible for this
inhibition, several other methylenedioxyphenyl compounds were examined for
their abilities to inhibit DEHP hydrolysis in vitro (Melancon and Lech
1979). The potent esterase inhibitor paraoxon also was used for comparison.
The hydrolysis of DEHP by serum and by a post-mitochondrial liver
fraction was inhibited substantially by paraoxon, PBO, and tropitol (Table
42). The remaining methylenedioxyphenyl compounds had little or no
inhibitory effect.
TABLE 42. EFFECT OF MICROSOMAL INHIBITORS ON DEHP HYDROLYSIS
Inhibitor
Control
Piperonyl butoxide
Saffrole
Tropitol
Piperonyl alcohol
1 , 3-benzodioxole
Piperonal
feroxon
Serum, d Liver homogenate
nmoles DEHP (8,000 g supernate),
metabolized nmoles DEHP metabolized
5 x 10~5 Af
1 x 10~3 M
5 x 10~5 M
1 x 10~3 M
5 x 10~5 M
1 x 10~3 M
5 x 10~5 M
\ x 10~3 M
5 x 10~5 M
1 x 10~3 M
5 x 10~5 M
1 x 10~3 M
5 x 10~5 M
1 x 10~3 M
1.76 ± 0.16b
0.62 ± 0.09C
0.09 ± 0.01°
1.72 ± 0.12
1.59 ± 0.22
0.68 ± 0.22°
0.06 ± 0.00°
2.23 ± 0.20
2.52 ± 0.15
1.93 ± 0.10
1.74 ± 0.10
1.93 ± 0.16
1.19 ± 0.17°
0.05 ± 0.01C
0.06 ± 0.01°
1.26 ± 0.14
0.33 ± 0.10C
0.13 ± 0.01C
1.24 ± 0.06
1.21 ± 0.28
0.43 ± 0.06C
0.16 ± 0.02C
1.40 i: 0.17
1.82 ± 0.13°
1.32 ± 0.08
1.16 ± 0.14
0.94 ± 0.07
1.39 ± 0.11
0.13 ± 0.00C
0.09 ± 0.01°
aOne ml of either a 1-5 dilution of trout serum in 0.010 M sodium phosphate
(pH 7.2) or the 8,000 g supernatant fraction of a 1-4 homogenate of liver
in 0.25 M sucrose diluted to the original homogenate volume were added to
1 ml of 0.01 M potassium phosphate (pH 7.2) containing 10 nmoles of C-
DEHP and the indicated inhibitor and incubated at 22°C for 1 h.
Data presented are the mean ± S.E. of observation from two experiments,
each of which contained three control incubations and two for each
inhibitor concentration.
cSignificantly different from control, p < 0.025; those not so labeled,
p > 0.05.
Additional experiments were performed on inhibition of the metabolism
of DEHP by microsomes and by cytosol. The number of inhibitors was reduced
by eliminating several examples of methylenedioxyphenyl compounds with short
chain substituents. The data (Table 43) show substantial inhibition of DEHP
120
-------
S
CO
M
O
S
a
PL,
a
W
Q
§
co
Oi
o
H
rH
CQ
M
a
H
i
5
CO
U
M
S
Pn
o
H
O
W
tn
fa
U
en
u
*^
3
H
i-H
O
CO
o
4-1
PL, ^3
a oi
W N
O -H
J
^1
CO O
01 -Q
rH CO
O 4J
§ e
co
0) *~^
° 0
S ss
g ^
PM ^
a oi
W N
Q iH
i-H
CO O
0) .0
i-H CO
O 4-1
6 B
CN
01
co S
oi C
rH 0
0 >4H
"
rH
01
CO 0
4H
PL,
a
s "S
sS
i-H ®
Q
a
cfl PLI T3
co «sa cu
§£gS
o e* -H
CO P CO O
O
CNJ
e
o
01
rH T3
gl
rl 4J
CU 3
IS"0
oo
o
o
-H
o
CD
vO
CN
CD
-H
en
vO
rH
VO
O
CD
-H
rH
CD
vO
rH
O
-H
,,-j.
o
i-H
O
CD
-H
m
CD
rH
CD
-H
vO
f*^
o
0
O)
o
^
0
•H
O
N
C
01
"f
en
i-H
y
CN
O
CD
-H
o
CD
y
rH
CD
-H
o
0
•
rH
O
rH
CD
•H
Oi
CM
CD
y
in
0
CD
-H
f-.
en
CD
0
CD
-H
en
en
CD
y
O
o
o
-H
m
o
^
o
a
o
o
CO
M
y
0
CD
-H
i-H
CD
y
oo
o
CD
-K
^
en
9
O
y
CD
^,
S
CD
y
in
o
CD
•tt
st
i-H
CD
y
CD
-W
in
H
CD
y
o
0
00
CN
(
0
rH
O
•H
O<
O
^^
CN
0)
a M
a. cu
01 ^~s
4-1
co a
O.Q
co <3
.§
ft %*
gen
•H O
CO rH
CO
cfl X
4-1
O CN
a,
si
rH *
O x-v
O CU
4-1
4H CO
0 U
•H
rH Ft^
s c
•H
rH
01
0 rl
4J 0»
CU ^-^
"O (-1
CO O
4J
CO -H
cd 4^
rH C
0 -H •
CO fi
0 ^
4J rH
r*?^
y 1 rl
0 0
(-1 rH Irl
O
0 O
C fri CN
o a CM
•H W
CO Q 4J
e i co
01 U
o«
3 4-1
co ;§; co
. jvf^ ^
§1-1 y
i a
O O iH
CO rH
O 01
rl X rl
y cu
•H m 15
&
60/->
<4H C "O
O -H 01
rH -H Cfl
S co y
4-1 iH
01 C T3
COS
o y -ri
eO
cu
••j
42
-o
01
e
•H
c
o
y
y
•H
"&
MH
O
42
y
cO
0)
CO
c
01
rl
01
a
CU
p
4-1
§
rl
<4H
CO
O
•H •
4J rl
Cfl O
> 4-1
rl ^H
01 43
CO -H
o c
•H
MH
O 43
y
• CO
U 01
•
CO IH
o
•H MH
C! O
cfl 3
CU 4J
rrj
01 C
43 CO
4-1
CO
a) q!
rl 0
CO -H
4J
T3 cO
01 43
^j 3
a u
0) C3
CO TH
0)
rl rH
a o
rl
Cfl 4->
CO O
a y
m
o
o
^
a
M
rH
O
(-1
4-1
C3
0
y
0
o
rl
>4H
4-1
C
01
rl
01
>4H
>4H
•rt
T3
rH
4J
13
y
iH
14H
•rl
C
00
•H
CO
y
CN
T3
C
cO
i — i
co
0)
4J
•H
i-H
O
Cfl
4-1
CU
e
t.
cfl
rH
a
OI
rl
ctf
CN
S
"c
cO
rH
121
-------
metabolism by every inhibitor in this set of experiments except
1,3-benzodioxole.
The results suggest that a fortuitous similarity in the structures of
DEHP, 2,4-DBE, and PBO is responsible for the inhibition of DEHP hydrolysis
by PBO.
EFFECT OF INDUCERS ON DISPOSITION OF ORGANIC CHEMICALS IN RAINBOW TROUT
The preceding study showed that PBO inhibited DEHP metabolism in vivo
and -in vitro* Numerous experiments described earlier in this report also
demonstrated that a number of chemicals induced hepatic cytochrome(s) P-450
and related enzyme activities in trout. The next step was to determine
whether the induction of these enzyme activities affected the metabolism and
disposition of xenobiotics by fish.
In our first experiment of this type, contrp]. trout and trout injected
2,3-benzanthracene wei
6 h (Statham et al. 1978).
with 2,3-benzanthracene were exposed to aqueous C-methylnaphthalene for
Increased rates of metabolism and elimination of C-2-methyl-
naphthalene in vivo were observed after pretreatment of the trout with 2,3-
benzanthracene. Figure 49 demonstrates that the rate of appearance of
radioactive material in bile was increased dramatically by pretreatment with
2,3-benzanthracene. Interestingly, the initial levels of C were higher in
livers of induced trout and appeared to be retained longer during the
washout period. Pretreatment with 2,3-benzanthracene did not appear to
affect significantly the rate of disappearance of radioactive material from
blood or muscle.
The TLC of extracts of bile indicated a greater proportion of polar
radioactive materials related to 2-methylnaphthalene and less parent
compound in bile after induction of monooxygenation by 2,3-benzanthracene
(Figure 50). Although the polar metabolites have not yet been characterized,
several different compounds appear to occur in the polar materials.
Subsequent experiments to investigate the effects of induction on the
disposition and metabolism of foreign chemicals by trout utilized a 24-h
exposure to the ^C-labeled chemical without an elimination period (Melancon
and Lech 1979).
Trout injected with g-naphthoflavone and vehicle-injected trout showed
several differences in tissue levels of ^C following exposure to ^C-
naphthalene or 1^C-2-methylnaphthalene (Table 44). Biliary levels of 14C in
3-naphthoflavone- (3NF) treated fish that had been exposed to 1^C-
naphthalene were 4.6 times those of control fish, while liver l^C was
slightly less than that of control fish and muscle and blood levels of C
were about half those found in control fish. The biliary C from control
and NF-treated fish consisted almost entirely of polar material while
the C present as polar material in muscle and liver from 3NF-treated trout
were 2.4 and 2.8 times the control, respectively. Biliary levels of C in
NF-treated fish exposed to C-2-inethylnaphthalene were 8.2 times those of
122
-------
EXPOSURE 14C-METHYLNAPHTHALENE 0.05mg/L
*
CO
z
y
$
3
LJ
LJ
-7
LJ _
M
£=>
50-
40-
30-
20-
BILE i
;
f ./
' /
/
LU
LJ
_l
<
5 e
^
UJ ^
Z !_
LJ O
_J
^1
h- \
X bo
^^
>•
I
UJ
3.0^
2.5-
2.0-
1.5-
1.0-
0.5^
-I- -I
CONTROL
BATREATED
LIVER
0 3
WASHOUT TIME(HRS)
Figure 49. Uptake and elimination of C-2-methylnaphthalene
derived material in rainbow trout. Each point is the mean
± S.E. (n = 3 in two separate experiments). *p < 0.05.
123
-------
UJ
z
UJ
LJ
cfl
4-J O
3 -H
O T3
rl C
4-1 -H
C CO
•H 4J
O
CO PL,
0) CO
4J
•H 13
rH Q)
O TJ
O O O O
in < ro CM
o
o o o o o o
in -3- n CM i—
0 nVIOl JO lN30d3d
17L
4-1 CO
0)
0)
QJ
r-l
0)
CO
cd
p,
r
4-1 n]
0) 00
0 H
I O
CM ^^
O O
LTl
•H iH
IH ••
O O
O
Q)
CD C
•H
T3
cd
rl
Q)
•H
,C
P- C8 rH
CS > cfl
rl '• rd
00 W 4-1
O O ,fl
4-1 j- a,
O •• r-l
a)
0
•H rH
O O
w g
00
•rl
Pn
124
-------
TABLE 44. EFFECT OF FRE-ADMINISTRATION OF -NAPHTHOFLAVONE ON THE
DISPOSITION AND METABOLISM OF 14C-LABELED CHEMICALS IN RAINBOW TROUT
Tissue
Tissue level of
parent chemical
+ metabolites
(H g/g or y g/ml)
Metabolites,
Tissue level of
parent chemical
+ metabolites
(yg/g or yg/ml)
Metabolites,
Biled
Muscle*
Liverb
Blooda
67.2 ± 5.1
2.25 ± 0.23
2.05 ± 0.12
1.83 ± 0.23
Naphthalene
98
5.1 ± 0.4
8.5 ± 0.5
308 ± 21
1.25 ± 0.16
1.72 ± 0.01
0.97 ± 0.08
99
12.3 ± 0.9
24.0 ± 1.8
Bilea
Muscle0
Liverc
Blood3
Bilea
Muscle0
Liver0
Blooda
150 ± 24
4.9
10.8
3.3 ± 0.2
14.7 ±0.8
575d
22d
2.01 ± 0.12
2-methylnaphthalene
96 1233 ± 201
2 2.6
10 5.0
1.9 ± 0.1
1,2,4-trichlorobenzene
65
0.8
3.7
87.5 ± 5.5
299d
42d
1.03 ± 0.04
100
10
40
98
2.1
6.2
Groups of eight trout were injected intraperitoneally with corn oil or a
solution of NF in corn oil (100 mg/ml) at a rate of 1 ml/kg. After 48 h,
groups of fish were exposed to one of the above chemicals for 24 h. The
water levels of the chemicals for control and induced trout were
naphthalene, 0.52 and 0.45 mg/liter; 2-methylnaphthalene, 0.28 and 0.36
mg/liter; and 1,2,4-trichlorobenzene, 0.20 and 0.20 mg/liter, respectively.
aAliquots of blood and bile from each fish were used to determine levels
of C. Values are the average ± S.E. Metabolite determinations utilized
pooled bile samples.
Each sample consisted of pooled muscle or liver from two fish. Thus four
samples per group were used to determine tissue C levels and percentage
of metabolites. Values are average ± S.E.
°Each sample consisted of pooled muscle or liver from all eight fish in the
group.
Tissue weights were not obtained. The total parent compound plus
metabolites which were extracted is given.
125
-------
control fish while C levels in muscle, liver, and blood were about half
those in control fish. The biliary C from both groups of fish represented
almost entirely polar material, while for liver and muscle the tissues from
3NF-treated trout contained respectively 4 and 5 times the percentages of
polar material found in these tissues from control trout.
The disposition of C-l,2,4-trichlorobenzene also was studied in NF-
injected and vehicle-injected trout. Biliary C was 6 times as great in
3NF-treated trout as in vehicle-injected fish. Levels of C in liver also
were great in NF-injected fish, while levels of C in muscle and blood
were lower. The percentages of C present as polar material in bile,
muscle, and liver were all higher in NF-treated trout than in vehicle-
injected trout.
The bile from control and NF-induced fish exposed to each of the
three C-labeled chemicals was examined by TLC (Figure 51, Figure 52, and
Figure 53). In all cases the bile from NF-treated fish had a greater
polarity than from control trout.
Earlier it was shown that PBO, an inhibitor of xenobiotic metabolizing
enzymes, affects the disposition and metabolism of ^C-di-2-
ethylhexylphthalate and -^C-PCA in rainbow trout. The experiments
demonstrate that pretreatment of trout with NF results in substantial
increases in the amounts of biliary metabolites of the three •*• ^C-labeled
chemicals. Substantial changes were observed in the tissue levels of C
accumulated from these chemicals and in the fraction of the C that was
present as metabolites.
Some aquatic pollutants, therefore, may be inducers or inhibitors of
the activities of hepatic microsomal monooxygenase enzymes in fish, thereby
affecting the metabolism and disposition of a variety of pollutants in these
fish. Reports by Rayne (1976) and Burns (1976) describe increased levels of
microsomal enzyme activities which appear to result from environmental
exposure to petroleum-derived pollutants. In order to evaluate whether such
induction is significant, in vivo studies must be conducted on the
disposition and metabolism of pollutants at the low levels that are found in
the aquatic environment.
126
-------
1000-1
500-
CONTROL
O
CD
UJO
§
-------
,C
P.
I
O
rs
-o
IAIO d3d lAldQ
S3in08Vi3IAI 4- 3N3HVHiHdVN-0J
'O
(U
en
o
3
o
VI
•8
•H
CD
"8
o
3
•a
oa
•a
cd
4J
o
o
o
CJ
J-
CO
•H
H
•rl
O CM
U H
CM
m
0)
M
3.
•H
128
-------
B NF - Treated
2
a.
a
0 2 4 « I 10
Distance from Origin (cm)
Figure 53. TLC of biliary 1IfC from control and 3NF-induced rainbow trout
exposed to 11(C-l,2,4-trichlorobenzene for 24 h. The mobile
phase was CHC13:MeOH:NH^OH (8:4:1). The solid oval area
represents the mobility of several trichlorophenols.
129
-------
SECTION 5
SIGNIFICANCE AND POTENTIAL APPLICATIONS OF THE RESEARCH
The biotransformation of foreign chemicals by fish results in the
presence of biotransformation products and the parent compound in fish
tissues. The rate of elimination of such biotransformation products from
fish tissues may be slower or more rapid than the rate of elimination of the
parent chemicals. These observations suggest that fish tissues should be
monitored for pollutants and their biotransformation products in order to
evaluate the presence of potentially harmful substances.
The results have demonstrated that biotransformation products of
certain chemicals accumulate in bile at much higher levels than that of the
parent in water, even several days after the exposure was terminated. It is
suggested that bile might be used for effective sampling in monitoring for
environmental spills, etc. In the case of pentachlorophenol the feasibility
of such monitoring was demonstrated by the results of a study concerning the
contamination of a small lake near Hattiesburg, Mississippi with
pentachlorophenol (Fate and Impact of Pentachlorophenol in a Freshwater
Ecosystem by Richard H. Pierce, Jr., sponsored by the U.S.-EPA, Athens,
Georgia). In that study, bile contained the highest level of
pentachlorophenol and metabolites of any tissue analyzed. In the case of
bass the bile level of pentachlorophenol and metabolites was thousands of
times the water level of pentachlorophenol.
It has been demonstrated that certain chemicals can affect fish liver
xenobiotic metabolizing enzymes and the biotransformation, disposition and
elimination of other chemicals -in vivo- Because of the wide variety of
pollutants which reach the aqueous environment, interactions such as these
should be anticipated from environmental pollutants.
130
-------
REFERENCES
Adamson, R.H. 1967. Drug metabolism in marine vertebrates. Fed. Proc.
26:1047.
Adamson, R.H., R.L. Dixon, F.L. Francis, and D. P. Rail. 1965. Comparative
biochemistry of drug metabolism by azo- and nitro-reductase. Proc.
Nat. Acad. Sci. 54:1386.
Addison, R.F., M.E. Zinch, and D.E. Willis. 1977. Mixed function oxidase
enzyme in trout (Salvelinus fontinalis) liver: Absence of induction
following feeding of P, p'-DDT, or p,p'-DDE. Comp. Biochem. Physiol.
75C, 39.
Albro, P.W., and B. Moore. 1974. Identification of the metabolites of
simple phthalate diesters in rat urine. Chromatog. 94:209.
Albro, B.W., R. Thomas, and L. Fishbein. 1973. Metabolism of diethylhexyl
phthalate by rats: Isolation and characterization of the urinary
metabolites. J. of Chrom. 76:32.
Alvares, A.P., D.R. Bickers, and A. Kappas. 1973. Polychlorinated
biphenyls: A new type of inducer of cytochrome P-448 in the liver.
Proc. Natl. Acad. Sci. U.S.A. 70:1321.
Alvares, A.P., and P. Siekevitz. 1973. Gel electrophoresis of partially
purified cytochrome P-450 from liver of variously pretreated rats.
Biochem. Biophys. Res. Communs. 54:923.
Ames, B.N. 1966. Assay of inorganic phosphate, total phosphate, and
phosphatases. In: Methods in enzymology, I.S.P. Colowick and N.O.
Kaplan, eds. Academic Press, New York. Vol. 8, p. 115.
Anders, M.W., and G.J. Mannering. 1966. Kinetics of the inhibition of the
#-demethylation of ethylmorphine by 2-diethylaminoethyl 2,2-diphenyl
valerate HC1 (SKF-SF5A) and related compounds. Mol. Pharmacol. 2:319.
Anderson, J.W. 1975. Laboratory studies on the effects of oil on marine
organisms: An overview. Am. Petroleum Inst. Publ. 4249:1.
Anderson, J.W., and J.M. Neff. 1975. Accumulation and release of petroleum
hydrocarbons by edible marine organisms. In: International symposium
on recent advances in the assessment of the health effects of
environmental pollutants. 3:1461. (World Health Organization).
131
-------
Augustinsson, K-B. 1959. Electrophoresis studies on bloodplasm esterases
II. Avian, reptilian, amphibian and piscine plasmata. Acta Chem.
Scand. 13:1089.
Bend, J.R., R.J. Bohl, and J.R. Fouts. 1973. Further studies of the
microsomal mixed function oxidase system of the little shake, Rasa.
eirinacea, including its response to some xenobiotics. Bull, lit* Desert
Island Biol. Lab. 13:9.
Bhavnagary, H.M., and S.M. Ahmed. 1973. A spectrophotometric method for
the micro-determination of piperonyl butoxide in the presence of
pyrethrins. Analyst 98:792.
Bickers, D.R., J. Eisman, A. Kappas, and A.P. Alvares. 1975. Microscope
immersion oils: Effects of skin application on cutaneous and hepatic
drug-metabolizing enzymes. Biochem. Riarmacol. 24:779.
Boobis, A.R., O.W. Nebert, and J.S. Felton. 1977. Comparison of 3-
naphthaflavone and 3-methylcholanthrene as inducers of hepatic
(cytachromets) P-448 and aryl hydrocarbon (benzofajpyrene) hydroxylase
activity. Mol. Pharmacol. 13:259.
Boylan, D.B., and B.W. Tripp. 1971. Determination of hydrocarbons in
seawater extracts of crude oil and crude oil fractions. Nature 230:44.
Branson, D.R., G.E. Blau, H.C. Alexander and W.B. Neely. 1975.
Bioconcentration of 2,2'4,4'-tetrachlorobiphenyl in rainbow trout as
measured by an accelerated test. Trans. Am. Fish. Soc. 104:785.
Brodie, B.B., and R.P. Maickel. 1962. Comparative biochemistry of drug
metabolism. Proc. 1st International Pharmacol. Meeting 6:299-324.
Buhler, D.R. 1966. Hepatic drug metabolism in fishes. Fed. Proc. 25:343.
Buhler, D.R., and M.E. Rasmusson. 1968. The oxidation of drugs by
fishes. Comp. Biochem. Physiol. 25:223.
Burke, M.D., and R.T. Mayer. 1974. Ethoxyresorufin: Direct fluorimetric
assay of a microsomal deethylation which is preferentially inducible by
3-methylcholanthrene. Drug Metab. Dis. 2:583.
Burns, K.A. 1976. Microsomal mixed function oxidases in an estuarine fish,
Fundulus heteroclitus, and their induction as a result of environmental
contamination. Comp. Biochem. Physiol. 53B:443.
Carlson, R.M. and R. Caple. 1976. Organo-chemical implications of water
chlorination. In: The environmental impact of water chlorination.
R.L. Jolly, ed. p. 73.
Carlson, R.M., and R. Caple. 1979. U.S. Environmental Protection Agency,
Coal Leachate Project, R 803952-01-0, Final report.
132
-------
Carter, J.E., D.B. Roll, and R.V. letersen. 1974. The in vitro hydrolysis
of di-(2-ethylhexyl)phthalate by rat tissues. Drug Metab. Dis. 2:341.
Casida, J.E., J.L. Engel. E.G. Essac, F.X. Kamienski, and S. Kuwatsuka.
1966. Methylene-C^-dioxyphenyl compounds: Metabolism in relation to
their synergistic action. Science 153:1130.
Cserjesi, A.J., and E.L. Johnson. Methylation of pentachlorphenol by
Triohoderma virgatum. Can. J. Microbiol. 18:45.
Daniel, J.W., and H. Bratt. 1974. Absorption, metabolism and tissue
distribution of di-(2-ethylhexyl) phthalate in rats. Toxicology 2:51.
deDuve, C., B.C. Pressman, R. Gianetto, R. Wattiaux, and F. Appelmans.
1955. Tissue fractionation studies b. Intracellular distribution
patterns of enzymes in rat-liver tissue. Biochem. J. 60:604.
DeFoe, D.L. 1975. A multichannel toxicant injection system for flow-
through bioassays. J. Fish. Res. Bd. Can. 32:544.
Dent, J.G., C.R. Elcombe, K.J. Netter, and J.E. Gibson. 1978. Rat hepatic
microsomal cytochrome(s) P-450 induced by polybrominated biphenyls.
Drug. Metab. Dis., 6:96.
Dent, J.G., K.T. Netter and J.E. Gibson. 1976a. Effects of chronic
administration of polybrominated biphenyls on parameters associated
with hepatic drug metabolism. Res. Comm. Chem. Pathol. Riarmacol.
13:75.
Dent, J.G., K.J. Netter, and J.E. Gibson. 1976b. The induction of hepatic
microsomal metabolism in rats following acute administration of a
mixture of polybrominated biphenyls. Toxicol. Appl. Riarmacol. 38:237.
Dent, J.G., U. Roes, K.T. Netter, and J.E. Gibson. 1977. Stimulation of
hepatic microsomal metabolism in mice by a mixture of polybrominated
biphenyls. J. Tox. Environ. Health 6:637.
Dewaide, J.H. 1971. Metabolism of xenobiotics, Leijn, Numegen.
Dewaide, J.H., and P.T. Henderson. 1968. Hepatic ff-dimethylation of
aminopyrine in rat and trout. Biochem. Riarmacol. 17:901.
Dungs, W., and E. Beryheim-Irps. 1973. A new methylation method for the
gas chromatography of barbituric acid. Anal. Lett. 6:185.
Dutton, G.J., and J. P. Montgomery. 1958. Glucuronide synthesis in fish and
the influence of temperature. Biochem. J. 70:17P.
Ecobiochon, D.J., and A.M. Comeau. 1974. Comparative effects of commercial
Aroclors on rat liver enzyme activities. Chem. Biol. Interactions
9:341.
133
-------
Elcombe, C.E., and J.J. Lech. 1973. Induction of monooxygenation in
rainbow trout by polybrominated biphenyls: A comparative study.
Environ. Health Perspec. 23:309-314.
Elcombe, C.E., and J.J. Lech. 1979. Induction and characterization of
hemoprotein(s) P-450 and monooxygenation in rainbow trout (Salmo
gairdneri). Toxicol. Appl. Pharmacol. 49:437.
Estabrook, R.W., J. Beterson, J. Baron and A.G. Hildebrandt. 1972. The
spectrophotometric measurement of turbid suspensions of cytochromes
associated with drug metabolism. In: Methods in pharmacology. C.F.
Chignell, ed. Appleton-Century-Crofts, New York. Vol. 2:303.
Fromm, P.O., and R.C. Hunter. 1969. Uptake of dieldrin by isolated
perfused gills of rainbow trout. J. Fish. Res. Bd. Can. 26:1939.
Fukami, J., T. Mitsui, K. Fujunaga, and T. Shishido. 1969. The selective
toxicity of rotenone between mammal, fish and insect. In: Biochemical
Toxicology of Insecticides, Academic Press, R.D. O'Brien, and J.
Yamamoto, eds. New York, p. 159.
Gianetto, R., and C. DeDuve. 1955. Tissue fractionation studies 4.
Comparative study of the binding of acid phosphatase, -glucuronidase
and cathepsin by rat-liver particles. Biochem. J. 59:433.
Glickman, A.H., C.N. Statham, A. Wu, and J.J. Lech. 1977. Studies on the
uptake, metabolism, and disposition of pentachlorophenol and
pentachloroanisole in rainbow trout. Toxicol. and Appl. Pharmacol.
41:649.
Goldstein, J.A., P. Hickman, V.W. Burse, and H. Bergman. 1975. A
comparative study of two polychlorinated biphenyl mixtures (Aroclors
1242 and 1016) containing 42% chlorine on induction of hepatic
porphyria and drug metabolizing enzymes. Toxicol. Appl. Pharmacol.
32:461.
Goldstein, J.A., P. Hickman, H. Bergman, J.D. McKinney, and M. P. Walker.
1977. Separation of pure polychlorinated biphenyl isomers into two
types of inducers on the basis of induction of cytochrome P-450 or P—
448. Chem. Biol. Interactions 17:69.
Green, D.E., S. Mii, and P.M. Kohout. 1955. Studies on the terminal
electron transport system 1. Succinic dehydrogenase. J. Biol. Chem.
217:551.
Guenthner, T.M., and G.J. Mannering. 1977. Induction of hepatic
monooxygenase systems of pregnant rats with phenobarbital and 3-
methylcholanthrene. Biochem. Pharmocol. 26:567.
Gutenmann, W.H., and D.J. Lisk. 1965. Conversion of 4-(2,4-DB) herbicide
to 2,4-D by bluegills. New York Fish Game J. 12:108.
134
-------
Hamelink, J.L., R.C. Waybrant, and R.C. Ball. 1971. A proposal: Exchange
equilibria control the degree chlorinated hydrocarbons are biologically
magnified in lentic environments. Trans. Am. Fish. Soc. 100:207.
Hansen, A.R., and J.R. Fouts. 1972. Some problems in Michaelis-Manten
kinetic analysis of benzpyrene hydroxylase in hepatic microsomes from
polycyclic hydrocarbon-pretreated animals. Chem. Biol. Interactions
5:167.
Hanson, L.G., I.P. Kapoor, and R.L. Metcalf. 1972. Biochemistry of
selective toxicity and biodegradability: Comparative 0-dealkylation by
aquatic organisms. Comp. Gen. Pharmacol. 3:339.
Kites, R.A., and K. Biemann. 1972. Water pollution: Organic compounds in
the Charles River, Boston. Science 178:158.
Hogan, J.W., and C.O. Rnowles. 1972. Metabolism of diazinon by fish liver
microsomes. Bull. Environ. Contain. Toxicol. 8:61.
Hodgson, E., and R. M. Philpot. 1974. Interaction of methylenedioxyphenyl
(1,3-benzodioxle) compounds with enzymes and their effect on mammals.
Drug Metab. Rev. 3:231-301.
Huang, K.C., and F.F. Collins. 1962. Conjugation and excretion of
aminobenzoic acid isomers in marine fishes. J. Cell. Comp. Physiol.
60:29.
Huang, Y.C., and R.G. Kemp. 1971. Properties of a phosphodiesterase with
high affinity for adenosine 3',5'-cyclic phosphate. Biochem. 10:2278.
Hubscher, G., and G.R. West. 1965. Specific assays of some phosphatases in
subcellular fractions of small intestinal mucosa. Nature 205:799.
Hunn, J.B., R.A. Schoettger, and W.A. Willford. 1968. Turnover and urinary
excretion of free and acetylated M.S. 222 by rainbow trout, Salmo
gairdneri. J. Fish Res. Bd. Can. 25:25.
Imai, Y., and R. Sato. 1966. Evidence for two forms of P-450 henoprotein
in microsomal membranes. Biochem. Biophys. Res. Commun. 23:5.
James, M.O., J.R. Fouts, and J.R. Bend. 1977. Xenobiotic metabolizing
enzymes in marine fish. In: Pesticides in aquatic environments,
Plenum Press, New York. p. 171.
Jolly, R.L., G. Jones, W.W. Pitt, and J.E. Thompson. 1976. Chlorination of
organics in cooling waters and process effluents. In: The
environmental impact of water chlorination. R.L. Jolly, ed. 115 p.
Kamienski, F.X., and S.D. Murphy. 1971. Biphasic effects of
methylenedioxphenyl synergists on the action of hexobarbital and
organophosphate insecticides in mice. Toxicol. Appl. Pharmacol.
18:833.
135
-------
Laemmli, U.K, and M. Favre. 1973. Maturation of the head of bacteriophage
T4.I DNA packaging event. J. Mol. Biol. 80:575.
Larson, R.A., and J.C. Weston. 1976. Analysis of water extracts of crude
petroleum by gel permeation chromatography. Bull. Environ. Contam.
Toxicol. 16:44.
Lech, J.J., and C.N. Statham. 1975. Role of gluronide formation in the
selective toxicity of 3-trifluoromethyl-4-nitrophenol (TFM) for the sea
lamprey: Compariative aspects of TFM uptake and conjugation in sea
lamprey and rainbow trout. Toxicol. Appl. Pharmacol. 31:150.
Lee, C.C., W.K. Craig, and P.J. Smith. 1974. Water-soluble hydrocarbons
from crude oil. Bull. Environ. Contam. Toxicol. 12:212.
Lee, R.F., R. Sauerheber, and G.H. Dobbs. 1972. Uptake, metabolism, and
discharge of polycyclic aromatic hydrocarbons by marine fish. Mar.
Biol. 17:201.
Levvy, G.A. 1952. The preparation and properties of $-glucuronidase 4.
Inhibition by sugar acids and their lactones. Biochem. J. 52:464.
Ljungquist, A., and K-B. Augustinsson. 1971. Purification and properties
of two carboxylesterases from rat-liver microsomes. Eur. J. Biochem.
23:303.
Lotliker, P., E.G. Miller, J.A. Miller, and J.E. Halver. 1967. Metabolism
of the carcinogen 2-acetylaminofluorene by rainbow trout. Proc. Soc.
Exp. Biol. Med. 124:160.
Macek, K.J., M.E. Barrow, R.F. Frasny, and B.H. Sleight, III. 1975.
Bioconcentration of ^C-pesticides by bluegill sunfish during
continuous aqueous exposure. In: Structure-Activity Correlations in
Studies of Toxicity and Bioconcentration with Aquatic Organisms. G.D.
Veith and D.E. Konasewich, eds. p. 119.
Maren, T.H., R. Embry, and L.E. Broder. 1968. The excretion of drugs
across the gill of the dogfish, Squalue acanthi-as• Comp. Biochem.
Physiol. 26:853.
Melancon, M.J. Jr., and J.J. Lech. 1976. Distribution and biliary
excretion products of di-2-ethylhexyl phthalate in rainbow trout. Drug
Metab. Dis. 4:112.
Melancon, M.J. Jr., and J.J. Lech. 1977. Metabolism of di-2-ethylhexyl
phthalate by subcellular fractions from rainbow trout liver. Drug
Metab. Dis. 5:29.
Melancon, M.J. Jr., and J.J. Lech. 1978. Distribution and elimination of
napthalene and 2-methylnapthalene in rainbow trout during short- and
long-term exposures. Arch. Environ. Contam. Toxicol. 7:207-220.
136
-------
Melancon, M.J. Jr., and J.J. Lech. 1979. Structural requirements for the
inhibition of phthalate ester hydrolysis in rainbow trout by
methylenediosyphenyl compounds. Xenobiotica 9:317.
Melancon, M.J. Jr., and J.J. Lech. 1979. Uptake, biotransformation,
disposition, and elimination of 2-methylnaphthalene and naphthalene in
several fish species. In: Aquatic Toxicology, ASTtl STP 667, L.L.
Marking and R.A. Kimerle, eds. Am. Soc. for Testing and Materials.
Melancon, M.J. Jr., J. Saybolt, and J.J. Lech. 1977. Effect of piperonyl
butoxide on disposition of di-2-ethylhexyl phthalate by rainbow
trout. Xenobiotica 7:633.
Montesano, R., A.J. Ingram, and P.N. Magee. 1973. Metabolism of
dimethylnitrosamine by amphibians and fish in vitro* Experientia
29:599.
Moore, R.W., and S.D. Aust. 1977. Induction of drug metabolizing enzymes
by 2,2', 4,4', 5,5'-hexabromobiphenyl. Pharmacol. 19:162.
Moore, R.W., S.D. Sleight, and S.D. Aust. 1978. Induction of liver
microsomal drug metabolizing enzymes by 2,2', 4,4', 5,5'-
hexabromobiphenyl. Toxicol. Appl. Riarmacol. 44(2):309-321.
Mount, D.I., and W.A. Brungs. 1967. A simplified dosing apparatus for fish
toxicology studies. Water Res. 1:21.
Murphy, S.D. 1966. Liver metabolism and toxicity of thiophosphate
insecticides in mammalian, avian and piscine species. Proc. Soc. Exp.
Biol. Med. 123:392.
Nakatsugawa, T., and P.A. Dahm. 1967. Microsomal metabolism and
parathion. Biochem. Pharmacol. 16:25.
Nakatsugawa, T., N.M. Tolman, and P.A. Dahm. 1969. Oxidative degradation
of diazinon by rat liver microsomes. Biochem. Pharmacol. 18:685.
Nazir, D.J., A.P. Alcaraz, B.A. Bierl, M. Beroza, and P.P. Nair. 1971.
Isolation, identification, and specific localization of di-2-ethylhexyl
phthalate in bovine heart muscle mitochondria. Biochem. 10:4228.
Neely, W.B., D.R. Branson, and G.E. Blau. 1974. Partition coefficient to
measure bioconcentration potential of organic chemicals in fish.
Environ. Sci. Technol. 8:1113.
Neff, J.M., B.A. Cox, D. Dixit, and J.W. Anderson. 1976. Accumulation and
release of petroleum-derived aromatic hydrocarbons by four species of
marine animals. Marine Biology 38:279.
-n, L.E., J.L. Allen, and J.W. Hogan. 1977. Biotransformation and
elimination of the herbicide, dinitramine, in carp. J. Agr. Food Chem.
25(3):554-556.
137
-------
Payne, J.F. 1975. Field evaluation of benzopyrene hydroxylase induction as
a monitor of marine petroleum pollution. Science 191:945.
Eayne, J.F., and W.R. Penrose. 1975. Induction of aryl hydrocarbon
(benzo[a]pyrene) hydroxylase in fish by petroleum. Bull. Environ.
Contam. Toxicol. 14:112.
Pederson, M.G., W.K. Hershberger, P.K. Zachariah, and M.R. Juchau. 1976.
Hepatic biotransformation of environmental xenobiotics in six strains
of rainbow trout (Salmo ga-irdneri"). J. Fish Res. Bd. Can. 33:66.
Potter, J.L., and R.D. O'Brien. 1964. Rarathion activation by livers of
aquatic and terrestial vertebrates. Science 144:55.
Pohl, R.J., J.R. Bend, A.M. Guarino, and J.R. Fouts. 1974. Hepatic
microsomal mixed-function oxidase activity of several marine species
from coastal Maine. Drug Metab. Dis. 2:545.
Poland, A., and E. Glover. 1977. Chlorinated biphenyl induction of aryl
hydrocarbon hydroxylase: A study of the structure activity
relationship. Mol. Pharmacol. 13:924.
Rodgers, C.A., and D.L. Stalling. 1972. Dynamics of an ester of 2,4-D in
organs of three fish species. Weed Sci. 20:101.
Ross, E., and G. Schatz. 1973. Assay of protein in the presence of high
concentrations of sulfhydryl compounds. Analyt. Biochem. 54:304.
Roubal, W.T., T.K. Collier, and D.C. Malins. 1977. Accumulation and
metabolism of carbon-14 labeled benzene, naphthalene, and anthracene by
young coho salmon (Oncorhynchue kistuch)• Arch. Environ. Contam.
Toxicol. 5:513-529.
Sanborn, H.R., and D.C. Malins. 1977. Toxicity and metabolism of
naphthalene: A Study with marine larval invertebrates. Proc. Soc.
Exp. Biol. Med. 154:151.
Sanborn, J.R., R.L. Metcalf, C-C. Yu, and P-Y. Lu. 1975. Plasticizers in
the environment: The fate of di-#-octylphthalate (DOP) in two model
ecosystems and uptake and metabolism of DOP by aquatic organisms.
Arch. Environ. Contam. Toxicol. 3:244.
Schenkman, J.D., H. Remmer, and R.W. Estabrook. 1967. Spectral studies of
drug interaction with hepatic microsomal cytochrome. Mol. Pharmacol.
3:113-123.
Schoettger, R.A. 1970. Toxicology of thiadan in several fish and aquatic
invertebrates. Inv. Fish Control 35:3.
Schultz, D. P. 1973. Dynamics of a salt of (2,4-dichlorophenoxy) acetic
acid in fish, water, and hydrosol. J. Agr. Food Chem. 21:185.
138
-------
Schwark, W.S., and D.J. Ecobichon. 1968. Subcellular localization and
drug-induced changes of rat liver and kidney esterases. J. Physiol.
Pharmacol. 46:207.
Shibko, S., and A.L. Tappel. 1964. Distribution of esterases in rat
liver. Arch. Biochem. Biophys. 106:259.
Sladek, N.E., and G.J. Mannering. 1966. Evidence for a new P-450
hemoprotein in hepatic microsomes from methylcholanthrene treated
rats. Biochem. Biophys. Res. Commun. 24:668.
Solyom, A., and E.G. Trams. 1972. Enzyme markers in characterization of
isolated plasma membranes. Enzyme 13:329.
Sottocasa, G.L., B. Kuylenstierna, L. Ernster, and A. Bergstrand. 1967. An
electron-transport system associated with the outer membrane of liver
mitochondria. J. Cell. Bio. 32:415.
Stalling, D.L., J.W. Hogan, and J.L. Johnson. 1973. Phthalate ester
residues - their metabolism and analysis in fish. Envir. Health
Berspec. 3:159.
Stanton, R.H., and M.A.Q. Khan. 1973. Mixed-function oxidase activity
toward cyclodiene insecticides in bass and bluegill sunfish. Bast.
Biochem. and Hiysiol. 3:354.
Statham, C.N., C.R. Elcombe, S.P. Szyjka, and J.J. Lech. 1978. Effect of
polycyclic aromatic hydrocarbons on hepatic microsomal enzymes and
disposition of methylnaphthalene in rainbow trout •in vivo* Xenobiotica
8:65-71.
Statham, C.N., and J.J. Lech. 1975. Metabolism of 2',5-dichloro-4'-
nitrosalicylanilide (Bayer 73) in rainbow trout. J. Fish Res. Bd. Can.
32:515.
Statham, C.N., and J.J. Lech. 1976. Studies on the mechanism of
potentiation of the acute toxicity of 2,4-D ff-butylester and 2',5-
dichloro-4'-nitrosalicylanilide in rainbow trout by carbaryl. Toxicol.
Appl. Hiarmacol. 36:281.
Statham, C.N., M.J. Melancon, Jr., and J.J. Lech. 1976. Bioconcentration
of xenobiotics in trout bile: A proposed monitoring aid for some
waterborne chemicals. Science 193:680.
Statham, C.N., S.P. Szyjka, L.A. Menahan, and J.J. Lech. 1977.
Fractionation and subcellular localization of marker enzymes in rainbow
trout liver. Biochem. Pharmacol. 26:1395.
Stegeman, J., R.L. Binder, and A.M. Orren. 1980. Hepatic and extrahepatic
microsomal electron transport components and mixed function oxidases in
the marine fish Stenotorms ber's-iaolor. Biochem. Riarmacol.
139
-------
Sweeley, C.C., W.H. Elliott, I. Fries, and R. Ryhage. 1966. Mass
spectrometric determination of unresolved components in gas
chromatographic effluents. Anal. Chem. 38:1549.
Tanaka, A., T. Adachi, T. Takahashi, and T. Yamaha. 1975. Biochemical
studies of phthalic esters I. Elimination, distribution and metabolism
of di-(2-ethylhexyl)phthalate in rats. Toxic. 4:253.
Ullrich, V., and P. Weber. 1972. The 0-dealkylation of 7-ethoxycoumarin by
liver microsomes. Hoppe-Seyler's Z. Physiol. Chem« 353:1171.
Underhay, E., S.J. Holt, H. Beaufay, and C. deDuve. 1956. Introcellular
localization of esterase in rat liver. J. Biophys. Biochem. Cytol.
2:635.
Wiebel, F.J., J.C. Leutz, L. Diamond, and H.V. Gelboin. 1971. Aryl
hydrocarbon (benzo[a]pyrene) hydroxylase in microsomes from rat
tissues: Differential inhibition and stimulation by benzoflavone and
organic solvents. Arch. Biochem. Biophys. 144:78.
Williams, D.T. 1973. Dibutyl- and di-(2-ethylhexyl)phthalate in fish.
Agr. Food Chem. 21:1128.
Williams, D.T., and B.J. Blanchfield. 1974. Retention, excretion and
metabolism of di-(2-ethylhexyl)phthalate administered orally to the
rat. Bull. Environ. Contain. Toxicol. 11:371.
Williams, D.T., and B.J. Blanchfield. 1975. The retention , distribution,
excretion, and metabolism of dibutyl phthalate-7-^C in the rat. Agr.
Food Chem. 23:854.
Yamamoto, I. 1970. Mode of action of pyrethroids, nicotinoids, and
rotenoids. Ann. Rev. Entomol. 15:257.
Yobs, A. R. 1972. Levels of polychlorinated biphenyls in adipose tissue of
the general population of the nation. Environ. Health Perspec. 1:79.
Zitko, V. 1977. The accumulation of polybrominated biphenyl by fish.
Bull. Environ. Contam. Toxicol. 17:285.
140
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing}
1. REPORT NO.
EPA-600/3-80-082
2.
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Uptake, Metabolism, and Disposition of Xenobiotic
Chemicals in Fish
Wisconsin Power Plant Impact Study
5. REPORT DATE
August 1980 Issuing Date
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
John Lech and Mark Melancon
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Pharmacology and Toxicology
Medical College of Wisconsin
Milwaukee, Wisconsin
10. PROGRAM ELEMENT NO.
1BA820
11. CONTRACT/GRANT NO.
R803971
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA/600/03
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The effects and fate in fish of a number of chemicals, including hydrocarbons and
chlorinated hydrocarbons, have been examined. The interactions between these chemicals
and fish have been studied using several approaches.
The uptake an elimination of lSC-labeled napthalene, 2-methylnapthalene, 1,2,4-tri-
chlorobenzene, pentachlorophenol, and pentachloroanisole were studied. Each of these
chemicals was taken up rapidly by rainbow trout. Increasing the duration of exposure to
C-napthalene or C-2-methylnapthalene affected the elimination of ^C-containing com-
ponents from these fish. Activities of cytochrome P-450-related xenobiotic metabolizing
enzymes in rainbow trout livers were induced. The quantities of biliary metabolites ^n
these fish were considerably higher than those found in non-induced trout.
Piperonyl butoxide reduced levels of biliary metabolites of pentachloranisole and
di-2-ethylhexyl-phthalate in trout and increased tissue levels of these chemicals." The
high levels of biotransformation products of these chemicals found in fish bile during
and after exposure to the chemicals in these studies support the possible use of bile
sampling in pollutant-modelling programs.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Xenobiotic chemicals
Uptake
Metabolism
Depuration
Fish
Coal-fired power plant
Fish
06/A
18. DISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (ThisReport)
Unclassified
21. NO. OF PAGES
157
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE
U.S. GOVERNMENT PRINTING OFFICE: 1980--657-165/0097
141
------- |